JP5296861B2 - Image acquisition device and focus method of image acquisition device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an image acquisition device and a focus method thereof, which can acquire a shift direction of a focal position in advance and can detect a focal position of a sample with high accuracy, with a simple structure. <P>SOLUTION: An image acquisition device M includes an optical path difference generation member 21 provided in a second light path L2. This can reduce light quantity when a second imaging device 20 captures an optical image (front focus) having a focal point in front of an optical image entering a first imaging device 18, and an optical image (rear focus) having a focal point behind that, and can secure light quantity when the first imaging device 18 captures an image. In the image acquisition device M, a position of a first imaging region 22A and a position of a second imaging region 22B in an imaging surface 20a of the second imaging device 20 are on a front side of a region 22C in which an optical image conjugated with a first optical image entering the first imaging device 18 enters, in a scanning direction of a sample S on the imaging surface 20a, thereby allowing a shift direction of a focal position of an object lens 15 to be acquired in advance with a simple structure. <P>COPYRIGHT: (C)2013,JPO&amp;INPIT

Description

  The present invention relates to an image acquisition device used for acquiring an image of a sample or the like and a focusing method thereof.

  As an image acquisition device, for example, there is a virtual microscope device that divides an imaging region of a sample into a plurality of regions in advance, images each divided region at a high magnification, and then combines them. In such image acquisition with a virtual microscope, conventionally, a focus map for the entire region of the sample is set as an imaging condition when acquiring an image of a sample such as a biological sample, and focus control based on the focus map is performed. Image acquisition of the sample is performed while performing.

  To create a focus map, first, an entire sample is acquired as a macro image using an image acquisition device including a macro optical system. Next, using the acquired macro image, the imaging range of the sample is set, the imaging range is divided into a plurality of divided areas, and a focus acquisition position is set for each divided area. After setting the focus acquisition position, the sample is transferred to an image acquisition apparatus having a micro optical system, the focus position at the set focus acquisition position is acquired, and a focus map is created from these focus positions.

  However, when creating such a focus map, there is a problem that processing takes time. Further, if the interval and the number of focal points to be acquired are suppressed, the time required for processing is shortened, but in this case, there is a problem that the focus accuracy is lowered. For this reason, development of a dynamic focus for acquiring a high-magnification image of a sample while acquiring a focal position is being promoted. This method is a light intensity difference or contrast difference between a light image (front pin) focused before a light image incident on an image acquisition device for image acquisition and a light image focused later (rear pin). Is a method of detecting the shift direction of the focal position with respect to the current height of the objective lens and acquiring the image by moving the objective lens in a direction to cancel the shift.

  For example, in the microscope system described in Patent Document 1, a second imaging unit that captures a region in front of the region captured by the first imaging unit, and an image captured by the second imaging unit are used for the first imaging unit. According to the automatic focusing control means for adjusting the in-focus position of the objective lens at the imaging position of the first imaging means and the distance between the divided areas and the moving speed of the sample, the divided areas are imaged by the second imaging means. Timing control means for aligning the timing of moving from the position to the imaging position of the first imaging means and the timing of positioning the imaging position of the divided area imaged by the second imaging means on the imaging surface of the first imaging means And are provided. For example, in the microscope apparatus described in Patent Documents 2 and 3, the optical path length difference in the light guide optical system for focus control is formed using a glass member.

JP 2011-081211 A Republished patent WO2005 / 114287 Republished patent WO2005 / 114293

  In the microscope system described in Patent Document 1 described above, an optical path difference optical system is formed by using a half mirror and a mirror, and lights having different optical path lengths are incident on the two imaging regions of the second imaging unit, respectively. ing. In this conventional microscope system, for example, a first imaging unit and a second imaging unit are configured by a line sensor. With a line sensor, the exposure time is short, so it is important to secure the amount of light in order to obtain a clear image.In contrast, with this conventional microscope system, the light is branched by the optical path difference optical system. There is a problem that it is difficult to secure.

  Further, in this conventional microscope system, by tilting the optical surface of the optical path branching unit, the region imaged by the second imaging unit is positioned in the sample scanning direction with respect to the region imaged by the first imaging unit. It is adjusted so as to be on the near side, and prior acquisition of the focal position shift direction is performed. However, with such a configuration, there is a problem that it is difficult to adjust the optical surface. Furthermore, since the optical path difference optical system is constituted by the half mirror and the mirror, there is a problem that the light passing through the optical path difference optical system is difficult to fit on the imaging surface of the second imaging means.

  The present invention has been made to solve the above-described problems, and provides an image acquisition apparatus and a focus method thereof capable of acquiring in advance the shift direction of the focus position with a simple configuration and accurately detecting the focus position of the sample. For the purpose.

  In order to solve the above problems, an image acquisition apparatus according to the present invention includes a stage on which a sample is placed, stage control means for scanning the stage at a predetermined speed, a light source for irradiating light toward the sample, and a sample A light guiding optical system including a light branching unit that branches the optical image of the first optical path into a first optical path for image acquisition and a second optical path for focus control, and a first optical image that is branched into the first optical path. A first imaging unit that acquires one image, a second imaging unit that can acquire a two-dimensional image of the second image by the second optical image branched into the second optical path, and a second image And a partial image of the second optical image is acquired on the imaging surface of the second imaging unit and a focus control unit that controls the focal position of imaging by the first imaging unit based on the analysis result. An area control means for setting the first imaging area and the second imaging area; An optical path difference generating member that generates an optical path difference in the second optical image along the in-plane direction of the surface, and the region control means is light conjugate with the first optical image incident on the first imaging means. The position of the first imaging region and the position of the second imaging region are set so as to be positioned on the front side in the scanning direction of the sample on the imaging surface with respect to the region where the image is incident.

  In this image acquisition device, since the optical path difference generating member is disposed in the second optical path, the first imaging area and the second imaging area of the second imaging means are incident on the first imaging means. A light image (front pin) focused before the light image and a light image (rear pin) focused later can be respectively captured. In this image acquisition device, since the optical path length difference can be formed without branching light in the second optical path for focus control, the amount of light to the second optical path necessary for obtaining information on the focal position is suppressed. Therefore, it is possible to secure a light amount when performing imaging with the first imaging means. In this image acquisition device, the position of the first imaging region and the position of the second imaging region on the imaging surface of the second imaging unit are conjugate with the first optical image incident on the first imaging unit. By positioning the front side of the scanning direction of the sample on the imaging surface with respect to the region where a simple light image is incident, the shift direction of the focal position can be acquired in advance with a simple configuration.

  The optical path difference generating member is a flat plate member arranged so as to overlap at least a part of the imaging surface, and the area control means is a flat plate so as to avoid the shadow of the second light image by the edge portion of the flat plate member. It is preferable to set the first imaging region and the second imaging region in a region overlapping the member and a region not overlapping the flat plate member, respectively. In this case, the configuration of the optical path difference generating member can be simplified by using the flat plate member. Further, since the edge portion of the flat plate member forms a shadow of the second optical image on the imaging surface of the second imaging device, the first imaging area and the second imaging area can be set by avoiding the shadow. The accuracy of control of the focal position can be ensured.

  Further, the optical path difference generating member is a member having a portion whose thickness continuously changes along the in-plane direction of the imaging surface, and the region control means is overlapped with a portion having a different thickness of the optical path difference generating member. It is preferable to set the first imaging region and the second imaging region. In this case, the distance between the front pin and the rear pin can be freely adjusted by adjusting the position of the first imaging region and the position of the second imaging region. As a result, the focal position of the sample can be detected with high accuracy.

  In addition, an objective lens that faces the sample and an objective lens control unit that relatively controls the position of the objective lens with respect to the sample based on control by the focus control unit, and the objective lens control unit is configured by the focus control unit. It is preferable not to drive the objective lens during execution of the focal position analysis and to move the objective lens in one direction with respect to the sample while the focal position analysis is not performed by the focus control means. In this case, since the positional relationship between the objective lens and the sample does not change during the analysis of the focal position, the analysis accuracy of the focal position can be ensured.

  In addition, the area control means is incident on the first imaging apparatus and the interval between the first imaging area and the area where the optical image conjugate with the first optical image incident on the first imaging apparatus is incident. It is preferable to set the position of the first imaging area and the position of the second imaging area so that the distance between the area where the optical image conjugate with the first optical image is incident and the second imaging area are different. Thereby, it is possible to sufficiently secure the exposure time in the first imaging region and the second imaging region.

  Further, the area control means is configured to change the imaging in the first imaging area from the imaging in the first imaging area based on the scanning speed of the stage and the interval between the first imaging area and the second imaging area. It is preferable to set a waiting time until imaging. Thereby, since the light from the same position of the sample is incident on the first imaging region and the second imaging region, the focal position can be controlled with high accuracy.

  The focus method of the image acquisition apparatus according to the present invention includes a stage on which a sample is placed, stage control means for scanning the stage at a predetermined speed, a light source that irradiates light toward the sample, and light from the sample. A light guide optical system including a light branching unit for branching an image into a first optical path for image acquisition and a second optical path for focus control; and a first optical image formed by a first optical image branched into the first optical path. A first imaging means for acquiring an image; a second imaging means capable of acquiring a two-dimensional image of the second image by the second optical image branched into the second optical path; and analyzing the second image. And a focus control means for controlling the focus position of the image pick-up by the first image pick-up means on the basis of the analysis result. A first imaging area and a second imaging area for acquiring a partial image of the two optical images. Then, an optical path difference generating member that generates an optical path difference in the second optical image along the in-plane direction of the imaging surface is disposed in the second optical path, and is incident on the first imaging means by the region control means. Setting the position of the first imaging area and the position of the second imaging area so as to be positioned on the front side in the scanning direction of the sample on the imaging surface with respect to the area where the optical image conjugate with the first optical image is incident. It is a feature.

  In the focusing method of the image acquisition device, the optical path difference generating member is disposed in the second optical path, so that the first imaging area and the second imaging area of the second imaging means are incident on the first imaging means. It is possible to capture a light image (front pin) focused before the light image to be performed and a light image (rear pin) focused later. In this focusing method, since the optical path length difference can be formed without branching light in the second optical path for focus control, the amount of light to the second optical path necessary for obtaining information on the focal position can be suppressed. It is possible to secure the amount of light when performing imaging with the first imaging means. Further, in this focusing method, the position of the first imaging region and the position of the second imaging region on the imaging surface of the second imaging unit are conjugate with the first optical image incident on the first imaging unit. By positioning the optical image on the front side in the scanning direction of the sample on the imaging surface with respect to the region on which the optical image is incident, the shift direction of the focal position can be acquired in advance with a simple configuration.

  Further, as the optical path difference generating member, a flat plate member arranged so as to overlap at least a part of the imaging surface is used, and the flat plate is used so as to avoid the shadow of the second light image by the edge portion of the flat plate member by the region control means. It is preferable to set the first imaging region and the second imaging region in a region overlapping the member and a region not overlapping the flat plate member, respectively. In this case, the configuration of the optical path difference generating member can be simplified by using the flat plate member. Further, since the edge portion of the flat plate member forms a shadow of the second optical image on the imaging surface of the second imaging device, the first imaging area and the second imaging area can be set by avoiding the shadow. The accuracy of control of the focal position can be ensured.

  Further, as the optical path difference generating member, a member having a portion whose thickness continuously changes along the in-plane direction of the imaging surface is used, and the region control unit is configured to overlap the different thickness portions of the optical path difference generating member. It is preferable to set the first imaging region and the second imaging region. In this case, the distance between the front pin and the rear pin can be freely adjusted by adjusting the position of the first imaging region and the position of the second imaging region. As a result, the focal position of the sample can be detected with high accuracy.

  The image acquisition apparatus includes an objective lens facing the sample, and an objective lens control unit that relatively controls the position of the objective lens with respect to the sample based on control by the focus control unit. Preferably, the objective lens is not driven during execution of the focus position analysis by the focus control means, and the objective lens is moved in one direction with respect to the sample while the focus position analysis by the focus control means is not executed. In this case, since the positional relationship between the objective lens and the sample does not change during the analysis of the focal position, the analysis accuracy of the focal position can be ensured.

  In addition, the region control unit causes the first imaging image to be incident on the first imaging region and the interval between the first imaging region and the first imaging region conjugate with the first optical image incident on the first imaging device. The position of the first imaging area and the position of the second imaging area are set so that the distance between the area where the optical image conjugate with the first optical image is incident and the second imaging area are different. It is preferable to do. Thereby, it is possible to sufficiently secure the exposure time in the first imaging region and the second imaging region.

  Further, the area control means changes the imaging in the first imaging area from the imaging in the first imaging area based on the scanning speed of the stage and the interval between the first imaging area and the second imaging area. It is preferable to set a waiting time until imaging. Thereby, since the light from the same position of the sample is incident on the first imaging region and the second imaging region, the focal position can be controlled with high accuracy.

  According to the present invention, the focal position shift direction can be acquired in advance with a simple configuration, and the focal position of the sample can be accurately detected.

It is a figure which shows one Embodiment of the macro image acquisition apparatus which comprises the image acquisition apparatus which concerns on this invention. It is a figure which shows one Embodiment of the micro image acquisition apparatus which comprises the image acquisition apparatus which concerns on this invention. It is a block diagram which shows the functional component of an image acquisition apparatus. It is a figure which shows the analysis result of the contrast value in case the distance to the surface of a sample corresponds with the focal distance of an objective lens. It is a figure which shows the analysis result of the contrast value in case the distance to the surface of a sample is longer than the focal distance of an objective lens. It is a figure which shows the analysis result of the contrast value in case the distance to the surface of a sample is shorter than the focal distance of an objective lens. It is a figure which shows an example of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows another example of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows another example of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows another example (forward direction) of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows another example (reverse direction) of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows another example (forward direction) of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows another example (reverse direction) of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows the modification of an optical path difference production | generation member. It is a figure which shows the relationship between the distance of the objective lens and the surface of a sample with respect to the scanning time of a stage. It is a figure which shows the control of the scanning direction of a stage by a stage control part. It is a figure which shows control of the scanning speed of a stage by a stage control part. It is a flowchart which shows operation | movement of an image acquisition apparatus.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of an image acquisition device and a focus method of the image acquisition device according to the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a diagram showing an embodiment of a macro image acquisition device constituting an image acquisition device according to the present invention. FIG. 2 is a diagram showing an embodiment of a micro image acquisition device constituting the image acquisition device according to the present invention. As shown in FIGS. 1 and 2, the image acquisition device M includes a macro image acquisition device M1 that acquires a macro image of the sample S and a micro image acquisition device M2 that acquires a micro image of the sample S. . The image acquisition apparatus M sets, for example, a plurality of line-shaped divided areas 40 (see FIG. 9) for the macro image acquired by the macro image acquisition apparatus M1, and each of the divided areas 40 is set to a high magnification by the micro image acquisition apparatus M2. This is a device that generates a virtual micro image by acquiring and combining them.

  As shown in FIG. 1, the macro image acquisition apparatus M1 includes a stage 1 on which a sample S is placed. The stage 1 is an XY stage that is driven in the horizontal direction by a motor or actuator such as a stepping motor (pulse motor) or a piezoelectric actuator. The sample S to be observed with the image acquisition device M is a biological sample such as a cell, for example, and is placed on the stage 1 in a state of being sealed with a slide glass. By driving the stage 1 in the XY plane, the imaging position with respect to the sample S can be moved. The stage 1 can reciprocate between the macro image acquisition device M1 and the micro image acquisition device M2, and has a function of transporting the sample S between both devices. The stage 1 may be provided in both the macro image acquisition device M1 and the micro image acquisition device M2.

  A light source 2 that irradiates light toward the sample S and a condenser lens 3 that condenses the light from the light source 2 onto the sample S are disposed on the bottom side of the stage 1. The light source 2 may be arranged so as to irradiate light obliquely toward the sample S. A light guide optical system 4 that guides a light image from the sample S and an imaging device 5 that captures a light image of the sample S are disposed on the upper surface side of the stage 1. The light guide optical system 4 includes an imaging lens 6 that forms an optical image from the sample S on the imaging surface of the imaging device 5. The imaging device 5 is an area sensor that can acquire a two-dimensional image, for example. The imaging device 5 acquires the entire image of the light image of the sample S incident on the imaging surface via the light guide optical system 4 and stores it in a virtual micro image storage unit 39 described later.

  As shown in FIG. 2, the micro image acquisition device M2 has a light source 12 and a condenser lens 13 similar to those of the macro image acquisition device M1 on the bottom surface side of the stage 1. A light guide optical system 14 that guides a light image from the sample S is disposed on the upper surface side of the stage 1. The optical system that irradiates the sample S with light from the light source 12 employs an excitation light irradiation optical system for irradiating the sample S with excitation light and a dark field illumination optical system for acquiring a dark field image of the sample S. May be.

  The light guide optical system 4 includes an objective lens 15 disposed so as to face the sample S, and a beam splitter (light branching means) 16 disposed at a subsequent stage of the objective lens 15. The objective lens 15 is provided with a motor or actuator such as a stepping motor (pulse motor) or a piezo actuator that drives the objective lens 15 in the Z direction orthogonal to the mounting surface of the stage 1. By changing the position of the objective lens 15 in the Z direction by these driving means, the focal position of the imaging in the image acquisition of the sample S can be adjusted. The focus position may be adjusted by changing the position of the stage 1 in the Z direction, or by changing the positions of both the objective lens 15 and the stage 1 in the Z direction.

  The beam splitter 16 is a part that branches the optical image of the sample S into a first optical path L1 for image acquisition and a second optical path L2 for focus control. The beam splitter 16 is disposed at an angle of about 45 degrees with respect to the optical axis from the light source 12. In FIG. 2, the optical path passing through the beam splitter 16 is the first optical path L1, and the beam splitter 16 The optical path reflected by 16 is the second optical path.

  In the first optical path L 1, an imaging lens 17 that forms an optical image (first optical image) of the sample S that has passed through the beam splitter 16, and an imaging surface is disposed at the imaging position of the imaging lens 17. A first imaging device (first imaging means) 18 is arranged. The first imaging device 18 is a device that can acquire a one-dimensional image (first image) based on a first light image of the sample S. For example, a two-dimensional CCD sensor capable of TDI (Time Delay Integration) driving, A line sensor is used. Further, if the method of sequentially acquiring the images of the sample S while controlling the stage 1 at a constant speed, the first imaging device 18 is a device that can acquire a two-dimensional image such as a CMOS sensor or a CCD sensor. There may be. The first images picked up by the first image pickup device 18 are sequentially stored in a temporary storage memory such as a lane buffer, and then compressed and output to an image generation unit 38 to be described later.

  On the other hand, in the second optical path L2, a field adjustment lens 19 for reducing the optical image (second optical image) of the sample reflected by the beam splitter 16, a second imaging device (second imaging means) 20, and Is arranged. In addition, an optical path difference generation member 21 that causes an optical path difference in the second optical image is disposed in the front stage of the second imaging device 20. The field adjustment lens 19 is preferably configured so that the second light image is formed on the second imaging device 20 with the same size as the first light image. Moreover, it is preferable that the optical path difference generating member 21 is arranged so that the surface facing the second imaging device 20 is parallel to the imaging surface (light receiving surface) 20a of the second imaging device. Thereby, refraction of light by the surface facing the second imaging device 20 can be reduced, and the amount of light received by the second imaging device 20 can be ensured.

  The second imaging device 20 is a device that can acquire a two-dimensional image (second image) based on the second optical image of the sample S, and for example, a CMOS (Complementary Metal Oxide Semiconductor), a CCD (Charge Coupled Device), or the like. These two-dimensional sensors are used.

  The imaging surface 20a of the second imaging device 20 is disposed so as to substantially coincide with the XZ plane orthogonal to the second optical path L2. A first imaging area 22A and a second imaging area 22B for acquiring a partial image of the second optical image are set on the imaging surface 20a (see, for example, FIG. 7). The first imaging region 22A and the second imaging region 22B are oriented in a direction perpendicular to the moving direction (scanning direction: Z direction) of the second optical image on the imaging surface 20a accompanying the scanning of the sample S. Is set. The first imaging area 22A and the second imaging area 22B are set with a predetermined interval, and both acquire a part of the second optical image in a line shape. Thereby, the optical image of the same area as the first optical image of the sample S acquired by the first imaging device 18 can be acquired as the second optical image in the first imaging area 22A and the second imaging area 22B. .

  Thereby, in the 2nd imaging device 20, before the 1st optical image which injects into the 1st imaging device 18 based on the position of 22 A of 1st imaging regions, and the position of 2nd imaging region 22B. A focused light image (front pin) and a later focused light image (rear pin) can be acquired. The focus difference between the front pin and the rear pin is incident on the thickness and refractive index of the optical path difference generating member 21 through which the second optical image incident on the first imaging region 22A passes, and on the second imaging region 22B. This depends on the difference between the thickness and the refractive index of the optical path difference generating member 21 through which the second optical image passes. An example of the shape of the optical path difference generation member 21 and the arrangement of the first imaging region 22A and the second imaging region 22B on the imaging surface 20a will be described later.

  FIG. 3 is a block diagram illustrating functional components of the image acquisition apparatus. As shown in the figure, the image acquisition apparatus M includes a computer system including a CPU, a memory, a communication interface, a storage unit such as a hard disk, an operation unit 31 such as a keyboard, a monitor 32, and the like. As constituent elements, a focus control unit 34, an area control unit 35, an objective lens control unit 36, a stage control unit 37, an image generation unit 38, and a virtual micro image storage unit 39 are provided.

  The focus control unit 34 is a part that analyzes the second image acquired by the second imaging device 20 and controls the focal position of imaging by the first imaging device 18 based on the analysis result. More specifically, the focus control unit 34, first, in the second imaging device 20, the contrast value of the image acquired in the first imaging region 22A and the contrast value of the image acquired in the second imaging region 22B. Find the difference between

  Here, as shown in FIG. 4, when the focal position of the objective lens 15 is in alignment with the surface of the sample S, the image contrast value of the front pin acquired in the first imaging region 22A and the second imaging region are obtained. After obtaining at 22B, the image contrast value of the pin substantially matches, and the difference value between them is almost zero.

  On the other hand, as shown in FIG. 5, when the distance to the surface of the sample S is longer than the focal length of the objective lens 15, the second imaging area is larger than the image contrast value of the front pin acquired in the first imaging area 22 </ b> A. After acquiring at 22B, the image contrast value of the pin becomes larger, and these difference values become positive. In this case, the focus control unit 34 outputs instruction information indicating that the objective lens 15 is driven in a direction approaching the sample S to the objective lens control unit 36.

  Further, as shown in FIG. 6, when the distance to the surface of the sample S is shorter than the focal length of the objective lens 15, the second imaging region is larger than the image contrast value of the front pin acquired in the first imaging region 22A. After the image acquisition at 22B, the image contrast value of the pin becomes smaller, and the difference value between these becomes negative. In this case, the focus control unit 34 outputs instruction information indicating that the objective lens 15 is driven in a direction away from the sample S to the objective lens control unit 36.

  The area control unit 35 is a part that controls the position of the first imaging area 22 </ b> A and the position of the second imaging area 22 </ b> B on the imaging surface 20 a of the second imaging device 20. The area control unit 35 first sets the first imaging area 22A at a preset position based on an operation from the operation unit 31, and after the imaging in the first imaging area 22A is performed, the first imaging area 22A is set. The setting of the imaging area 22A is canceled. Next, the second imaging area 22B is set with a predetermined interval from the first imaging area 22A in the Z direction (scanning direction), and after the imaging in the second imaging area 22B is performed, the second imaging is performed. The setting of the area 22B is cancelled.

  At this time, the waiting time W from the imaging in the first imaging area 22A to the imaging in the second imaging area 22B is an interval d between the first imaging area 22A and the second imaging area 22B, and It is set based on the scanning speed v of the stage 1. For example, if the waiting time W is a time W1 from the start of imaging in the first imaging area 22A to the start of imaging in the second imaging area 22B, the exposure time el of imaging in the first imaging area 22A, the first In consideration of the time st from the cancellation of the setting of the imaging region 22A to the setting of the second imaging region 22B, W1 = d / v-el-st can be obtained.

  Further, when the waiting time W is a waiting time W2 from the completion of imaging in the first imaging area 22A to the start of imaging in the second imaging area 22B, the second setting after the setting of the first imaging area 22A is cancelled. In consideration of the time st until the imaging region 22B is set, W2 = d / v-st. Further, the distance d between the first imaging region 22A and the second imaging region 22B is set based on the optical path length difference generated by the optical path difference generating member 21. However, this interval d actually corresponds to the distance on the slide of the sample S, and it is finally necessary to convert the interval d into the number of pixels in the second imaging region 22B. When the pixel size of the second imaging device 20 is AFpsz and the magnification is AFmag, the number of pixels dpix corresponding to the interval d is obtained by dpix = d ÷ (AFpsz / AFmag).

  Further, the area control unit 35 conjugates with the first optical image incident on the first imaging device 18 in setting the position of the first imaging area 22A and the position of the second imaging area 22B on the imaging surface 20a. The position of the first imaging area 22A and the position of the second imaging area 22B are set so as to be positioned on the near side (upper side) in the scanning direction of the sample S on the imaging surface 20a with respect to the area where a simple light image is incident To do. That is, the area control unit 35 captures images on the sample S in the first imaging device 18 such that the imaging positions on the sample S in the first imaging region 22A and the second imaging region 22B of the second imaging device 20 are the same. The position of the first imaging area 22A and the position of the second imaging area 22B are set so as to be on the near side of the position (position where the first imaging device 18 will capture images from now on) (see FIG. 17A). ).

  By such setting of the position of the first imaging region 22A and the position of the second imaging region 22B, as shown in FIG. 7, it is conjugate with the first optical image incident on the first imaging device 18. The distance between the region 22C where the optical image is incident and the first imaging region 22A, and the region 22C and the second imaging where the optical image conjugate with the first optical image incident on the first imaging device 18 is incident. The interval between the regions 22B will be different. For this reason, it is possible to secure a sufficient exposure time in the first imaging region 22A and the second imaging region 22B.

  The setting of the first imaging region 22A and the second imaging region 22B is appropriately executed according to the shape of the optical path difference generating member 21. For example, in the example shown in FIG. 7, the optical path difference generating member 21A formed of a flat glass member is arranged so as to overlap with the lower half region in the Z direction on the imaging surface 20a. In the imaging surface 20a, a region 22C where a light image conjugate with the first optical image incident on the first imaging device 18 is located at a substantially central portion of the lower half region of the imaging surface 20a. As the sample S is scanned by the stage 1, the sample S moves in the + Z direction in the field of view of the imaging surface 20a.

  The first imaging area 22A and the second imaging area 22B are both set on the −Z direction side with respect to the area 22C, and the first imaging area 22A does not overlap the optical path difference generation member 21A. The second imaging area 22B is set as the lower half area of the imaging surface 20a that overlaps the optical path difference generating member 21A. Note that the edge portion E of the optical path difference generating member 21A may form a shadow 23 of the second optical image on the imaging surface 20a. Therefore, it is preferable to set the first imaging region 22A and the second imaging region 22B at positions avoiding the shadow 23.

  For example, in the example shown in FIG. 8, an optical path difference generating member in which a flat glass member having substantially the same area as the imaging surface 20 a and a flat glass member having the same area as the lower half region of the imaging surface 20 a are overlapped. 21B is disposed so as to overlap the imaging surface 20a. The first imaging area 22A and the second imaging area 22B are both set on the −Z direction side with respect to the area 22C, and the first imaging area 22A has a thickness of one glass member. The second imaging area 22B is set in the lower half area of the imaging surface 20a that overlaps the optical path difference generation member 21B with a thickness of two glass members. Is set.

  Also in this case, the edge portion E of the optical path difference generating member 21B may form a shadow 23 of the second optical image on the imaging surface 20a. Therefore, it is preferable to set the first imaging region 22A and the second imaging region 22B at positions avoiding the shadow 23.

  For example, in the example shown in FIG. 9, the optical path difference generating member 21 </ b> C made of a prism-like glass member whose thickness continuously increases in the + Z direction is arranged so as to overlap the imaging surface 20 a. Then, a region 22C where a light image conjugate with the first light image incident on the first imaging device 18 is located at a substantially central portion of the imaging surface 20a, and the first imaging region 22A along the + Z direction. The second imaging area 22B and the area 22C are set in this order.

  In this case, the optical path difference through which the second light image incident on the first imaging region 22A passes by adjusting the position of the first imaging region 22A and the position of the second imaging region 22B with respect to the Z direction. The thickness of the generation member 21C and the thickness of the optical path difference generation member 21C through which the second light image incident on the second imaging region 22B passes can be changed. Thereby, the focus difference between the front pin and the rear pin can be adjusted. In addition, it is necessary to consider that the focus difference also changes depending on the refractive index difference in the optical path difference generating member 21.

  Further, if the optical path difference generating member 21 is arranged so that the thickness is symmetric with respect to the Z direction, the position of the first imaging region 22A and the second imaging region even when the scanning direction of the sample S is reversed. Bidirectional scanning of the sample S can be executed by changing the position of 22B to a position that is symmetric with respect to the region 22C. For example, in the example shown in FIGS. 10 and 11, a flat plate-shaped optical path difference generating member 21D similar to that in FIG. 7 is arranged so as to coincide with the center of the imaging surface 20a in the Z direction. In addition, the region 22C where the optical image conjugate with the first optical image incident on the first imaging device 18 is located so as to coincide with the center in the Z direction on the imaging surface 20a.

  As shown in FIG. 10, when the sample S is scanned in the + Z direction, in the upper half area of the imaging surface 20a, the first imaging area 22A is set to an area that does not overlap with the optical path difference generating member 21D. Then, the second imaging region 22B is set to a region overlapping with the optical path difference generation member 21D. As shown in FIG. 11, when the sample S is scanned in the -Z direction, in the lower half region of the imaging surface 20a, the first imaging region 22A is an area that does not overlap with the optical path difference generating member 21D. The second imaging area 22B is set to an area overlapping the optical path difference generating member 21D.

  In the example shown in FIGS. 12 and 13, the optical path difference generating member 21E made of a prism-shaped glass member whose thickness continuously increases toward the center in the Z direction coincides with the center in the Z direction of the imaging surface 20a. Are arranged as follows. In addition, the region 22C where the optical image conjugate with the first optical image incident on the first imaging device 18 is located so as to coincide with the center in the Z direction on the imaging surface 20a.

  Then, as shown in FIG. 12, when the sample S is scanned in the + Z direction, in the upper half area of the imaging surface 20a, the first imaging area 22A is on the upper side, and the second imaging area 22B is on the lower side. Is set to be As shown in FIG. 13, when the sample S is scanned in the −Z direction, in the lower half area of the imaging surface 20a, the first imaging area 22A is on the lower side, and the second imaging area 22B is on the upper side. Is set to be

  For example, as shown in FIG. 14A, the optical path difference generating member 21 in the case of performing bidirectional scanning of the sample S has a constant thickness in the center portion in the Z direction and both end portions in the Z direction outward. For example, as shown in FIG. 14B, an optical path difference generating member 21G made of two glass members having a prism shape with a right-angled triangle cross section may be used. You may arrange | position so that thickness may reduce toward the center side from the both ends part side of the surface 20a in the Z direction.

  When setting the position of the region 22C where the optical image conjugate with the first optical image incident on the first imaging device 18 is set on the imaging surface 20a, first, the distance of the objective lens 15 to the stage 1 is fixed. Then, the position of the stage 1 is adjusted so that the cross line of the calibration slide is positioned at the center of the visual field of the first imaging device 18. Next, the back focus of the second imaging device 20 is adjusted so that the cross line of the calibration slide falls within the field of view of the second imaging device 20. Finally, the position in the in-plane direction of the second imaging device 20 is adjusted so that the cross line of the calibration slide comes to a desired position on the imaging surface 20a of the second imaging device 20.

  The objective lens control unit 36 is a part that controls driving of the objective lens 15. Upon receiving the instruction information output from the focus control unit 34, the objective lens control unit 36 drives the objective lens 15 in the Z direction according to the content of the instruction information. Thereby, the focal position of the objective lens 15 with respect to the sample S is adjusted.

  The objective lens control unit 36 does not drive the objective lens 15 during the analysis of the focal position by the focus control unit 34, and moves the objective lens 15 in the Z direction until the analysis of the next focal position is started. Drive in only one direction along. FIG. 15 is a diagram illustrating a relationship between the distance between the objective lens and the surface of the sample with respect to the scanning time of the stage. As shown in the figure, during the scan of the sample S, the focal position analysis period A and the objective lens driving period B based on the analysis result occur alternately.

  The stage control unit 37 is a part that controls driving of the stage 1. More specifically, the stage control unit 37 scans the stage 1 on which the sample S is placed at a predetermined speed based on an operation from the operation unit 31. The scanning of the stage 1 relatively sequentially moves the imaging field of the sample S in the first imaging device 18 and the second imaging device 20. As shown in FIG. 16A, the scanning direction of the stage 1 is set so that the position of the stage 1 is returned to the scanning start position every time scanning of one divided region 40 is completed, and then the next divided region 40 is set in the same direction. As shown in FIG. 16B, after the scanning of one divided region 40 is completed, the stage 1 is moved in a direction orthogonal to the scanning direction to move to the next divided region. Bidirectional scanning may be performed by scanning 40 in the opposite direction.

  Further, although the scanning speed of the stage 1 during image acquisition is constant, there is actually a period in which the scanning speed is unstable due to the influence of vibration of the stage 1 immediately after the start of scanning. For this reason, as shown in FIG. 17, a scanning width longer than that of the divided region 40 is set, the acceleration period C in which the stage 1 accelerates, the stabilization period D until the scanning speed of the stage 1 stabilizes, and the stage 1 It is preferable that each of the deceleration periods F in which the deceleration occurs occurs when scanning outside the divided region 40. Thereby, it is possible to acquire an image in accordance with a constant speed period E in which the scanning speed of the stage 1 is constant. Note that imaging may be started during the stabilization period D, and the data portion acquired during the stabilization period D after image acquisition may be deleted. Such a method is suitable when using an imaging device that requires idle reading of data.

  The image generation unit 38 is a part that combines the acquired images to generate a virtual micro image. The image generation unit 38 sequentially receives the first image output from the first imaging device 18, that is, the image of each divided region 40, and synthesizes them to synthesize the entire image of the sample S. Then, an image having a lower resolution than this is created based on the synthesized image, and the high resolution image and the low resolution image are associated with each other and stored in the virtual micro image storage unit 39. In the virtual micro image storage unit 39, an image acquired by the macro image acquisition device M1 may be further associated. The virtual micro image may be stored as one image or may be stored as an image divided into a plurality of images.

  Next, the operation of the image acquisition device M described above will be described.

  FIG. 18 is a flowchart showing the operation of the image acquisition apparatus M. As shown in the figure, in the image acquisition device M, first, the macro image of the sample S is acquired by the macro image acquisition device M1 (step S01). The acquired macro image is binarized using, for example, a predetermined threshold, and then displayed on the monitor 32, and a micro image is selected from the macro image by automatic setting using a predetermined program or manual setting by an operator. A range to be acquired is set (step S02).

  Next, the sample S is transferred to the micro image acquisition device M2 side, and focus acquisition conditions are set (step S03). Here, as described above, imaging in the second imaging region 22B is started based on the scanning speed v of the stage 1 and the interval d between the first imaging region 22A and the second imaging region 22B. Set the waiting time W until it is done. More preferably, the exposure time el of imaging in the first imaging area 22A, the time st from when the setting of the first imaging area 22A is canceled until the second imaging area 22B is set, and the like are taken into consideration.

  After setting the focus acquisition conditions, scanning of the stage 1 is started, and micro images are acquired for each divided region 40 of the sample S by the micro image acquisition device M2 (step S04). When the micro image is acquired by the first imaging device 18, the second imaging device 20 uses the first imaging region 22A and the second imaging region 22B to obtain the difference between the contrast value of the front pin and the contrast value of the rear pin. Based on this, the deviation amount of the objective lens 15 with respect to the sample S is analyzed, and the position of the objective lens 15 is adjusted in real time. After the acquisition of the micro images for all the divided regions 40 is completed, the acquired micro images are synthesized and a virtual micro image is generated (step S05).

  As described above, in the image acquisition device M, the first imaging region 22A and the second imaging region of the second imaging device 20 are provided by arranging the optical path difference generation member 21 in the second optical path L2. In 22B, a light image (front pin) focused before the light image incident on the first imaging device 18 and a light image (rear pin) focused later can be respectively captured. In this image acquisition device M, an optical path length difference can be formed without branching light in the second optical path L2 for focus control, so that the second optical path L2 necessary for obtaining focal position information is obtained. The amount of light when the first imaging device 18 performs imaging can be secured sufficiently.

  Further, in this image acquisition device M, the first imaging device 18 enters the first imaging device 18 with the position of the first imaging region 22A and the position of the second imaging region 22B on the imaging surface 20a of the second imaging device 20. It is positioned on the front side in the scanning direction of the sample S on the imaging surface 20a with respect to the region 22C where the optical image conjugate with the first optical image is incident. Thereby, since the imaging result in the 2nd imaging device 20 can be acquired before the imaging timing in the 1st imaging device 18, the shift direction of the focal position of the objective lens 15 can be acquired in advance with a simple configuration. .

  Further, in this image acquisition device M, the imaging in the first imaging region 22A is completed based on the scanning speed v of the stage and the interval d between the first imaging region 22A and the second imaging region 22B. Is set to the waiting time W until the start of imaging in the second imaging area 22B. Therefore, the exposure time in the second imaging device 20 is sufficiently secured, and the focus position of the objective lens 15 can be controlled with high accuracy.

  When the optical path difference generating member 21 made of a flat glass member is used as the optical path difference generating member of the present embodiment, the configuration of the optical path difference generating member can be simplified. In this case, since the edge portion E of the flat plate member forms a shadow 23 of the second optical image on the imaging surface 20a of the second imaging device 20, the first imaging region 22A and the second imaging region 22A are avoided by avoiding the shadow 23. By setting the imaging region 22B, it is possible to ensure the accuracy of control of the focal position of the objective lens 15.

  When the optical path difference generating member 21 made of a glass member having a portion whose thickness changes along the in-plane direction of the imaging surface 20a in the second imaging device 20 is used as the optical path difference generating member of the present embodiment, the region By adjusting the position of the first imaging area 22A and the position of the second imaging area 22B by the control unit 35, the distance between the front pin and the rear pin can be freely adjusted. Thereby, for example, when there are a plurality of contrast peaks of the image captured by the second imaging device 20 or when the peak shape is flat, the focus of the sample S is adjusted by adjusting the focus difference between the front pin and the rear pin. The position can be detected with high accuracy.

  In the above-described embodiment, an apparatus for generating a virtual micro image has been exemplified. However, the image acquisition apparatus according to the present invention may be various as long as it acquires an image while scanning a sample at a predetermined speed with a stage or the like. It can be applied to the device.

DESCRIPTION OF SYMBOLS 1 ... Stage, 12 ... Light source, 14 ... Light guide optical system, 15 ... Objective lens, 16 ... Beam splitter (light branching means), 18 ... 1st imaging device (1st imaging means), 20 ... 2nd Imaging device (second imaging means), 20a ... imaging surface, 21 (21A to 21G) ... optical path difference generating member, 22A ... first imaging area, 22B ... second imaging area, 22C ... first optical image A region where a light image conjugate with the light is incident, 23... Shadow, 34... Focus control unit (focus control unit), 35 ... region control unit (region control unit), 36 .. objective lens control unit (objective lens control unit), E ... an edge portion, L1 ... first optical path, L2 ... second optical path, M ... image acquisition device, M1 ... macro image acquisition device, M2 ... micro image acquisition device, S ... sample.

Claims (12)

A stage on which the sample is placed;
Stage control means for scanning the stage at a predetermined speed;
A light source that emits light toward the sample;
A light guide optical system including a light branching unit that branches the optical image of the sample into a first optical path for image acquisition and a second optical path for focus control;
First imaging means for acquiring a first image by a first optical image branched into the first optical path;
A second imaging means capable of acquiring a two-dimensional image of the second image by the second optical image branched into the second optical path;
A focus control unit that analyzes the second image and controls a focal position of imaging by the first imaging unit based on the analysis result;
Area control means for setting a first imaging area and a second imaging area for acquiring a partial image of the second optical image on an imaging surface of the second imaging means;
An optical path difference generating member that is disposed in the second optical path and generates an optical path difference in the second optical image along an in-plane direction of the imaging surface;
The region control means is positioned on the imaging surface on the near side in the scanning direction of the sample with respect to a region where a light image conjugate with the first optical image incident on the first imaging means is incident. An image acquisition apparatus that sets a position of the first imaging region and a position of the second imaging region. The optical path difference generating member is a flat plate member arranged to overlap at least part of the imaging surface,
The area control means includes the first imaging area and the area that overlaps the flat plate member and the area that does not overlap the flat plate member so as to avoid the shadow of the second light image by the edge portion of the flat plate member. The image acquisition apparatus according to claim 1, wherein the second imaging area is set. The optical path difference generating member is a member having a portion whose thickness continuously changes along the in-plane direction of the imaging surface,
The image acquisition apparatus according to claim 1, wherein the area control unit sets the first imaging area and the second imaging area so as to overlap portions having different thicknesses of the optical path difference generation member. . An objective lens facing the sample;
An objective lens control means for controlling the position of the objective lens relative to the sample based on the control by the focus control means;
The objective lens control unit does not drive the objective lens during analysis of the focus position by the focus control means, and the objective lens is not applied to the sample while the focus position analysis is not executed by the focus control means. The image acquisition apparatus according to claim 1, wherein the image acquisition apparatus is moved in one direction.   The area control means includes an interval between an area where a light image conjugate with a first optical image incident on the first imaging apparatus and the first imaging area, and an interval between the first imaging apparatus and the first imaging apparatus. The position of the first imaging area and the position of the second imaging area are set so that the distance between the area where the incident first light image and the conjugate light image are incident and the second imaging area are different. The image acquisition apparatus according to claim 1, wherein the image acquisition apparatus is an image acquisition apparatus.   The area control means is configured to perform the second imaging from the imaging in the first imaging area based on the scanning speed of the stage and the interval between the first imaging area and the second imaging area. The image acquisition apparatus according to claim 1, wherein a waiting time until imaging in the imaging area is set. A stage on which the sample is placed;
Stage control means for scanning the stage at a predetermined speed;
A light source that emits light toward the sample;
A light guide optical system including a light branching unit that branches the optical image of the sample into a first optical path for image acquisition and a second optical path for focus control;
First imaging means for acquiring a first image by a first optical image branched into the first optical path;
A second imaging means capable of acquiring a two-dimensional image of the second image by the second optical image branched into the second optical path;
A focus control unit that analyzes the second image and controls a focus position of imaging by the first imaging unit based on a result of the analysis.
A first imaging area and a second imaging area for acquiring a partial image of the second optical image are set on the imaging surface of the second imaging means;
An optical path difference generating member that causes an optical path difference in the second optical image along the in-plane direction of the imaging surface is disposed in the second optical path,
The region control means is arranged to be positioned on the front side in the scanning direction of the sample on the imaging surface with respect to a region where a light image conjugate with the first optical image incident on the first imaging means is incident. A focus method for an image acquisition apparatus, wherein a position of a first imaging area and a position of the second imaging area are set. As the optical path difference generation member, using a flat plate member arranged to overlap at least a part of the imaging surface,
In order to avoid the shadow of the second light image by the edge portion of the flat plate member by the region control means, the first imaging region and the region that overlaps the flat plate member and a region that does not overlap the flat plate member The focus method of the image acquisition apparatus according to claim 7, wherein the second imaging area is set. As the optical path difference generating member, a member having a portion whose thickness continuously changes along the in-plane direction of the imaging surface,
The image acquisition apparatus according to claim 7, wherein the area control unit sets the first imaging area and the second imaging area so as to overlap portions having different thicknesses of the optical path difference generation member. Focus method. The image acquisition device includes:
An objective lens facing the sample;
An objective lens control means for controlling the position of the objective lens relative to the sample based on the control by the focus control means;
The objective lens control means does not drive the objective lens during execution of the focus position analysis by the focus control means, and the objective lens is not applied to the sample while the focus position analysis is not executed by the focus control means. The focus method for an image acquisition apparatus according to claim 7, wherein the focus method is moved in one direction.   The region control means causes the first imaging device to receive an interval between the first imaging region and a region where a light image conjugate with the first optical image incident on the first imaging device is incident on the first imaging device. The position of the first imaging area and the position of the second imaging area are set so that the distance between the area where the incident first light image and the conjugate light image are incident and the second imaging area are different. The focus method of the image acquisition apparatus according to claim 7, wherein the focus method is an image acquisition apparatus.   Based on the scanning speed of the stage and the interval between the first imaging area and the second imaging area, the area control means performs the second imaging from the imaging in the first imaging area. The focus method of the image acquisition apparatus according to claim 7, wherein a waiting time until the start of imaging in imaging is set.

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