JP2013127581A - Image acquisition device and focus method for image acquisition device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an imaging acquisition device able to obtain the deviating direction of a focal point under the same condition regardless of the direction of scan of a sample and to provide a focus method for the device.SOLUTION: The image acquisition device comprises a light guide optical system 14 including light splitting means 16 for splitting a light image of a sample S into two optical paths; first imaging means 18 split for the first optical path; second imaging means 20 split for the second optical path; focus control means for controlling the focused position of an image picked up by the first imaging means; area control means for setting an imaging area on an imaging surface of the second imaging means; and an optical path difference generating member 21 arranged in the second optical path and used to cause an optical path difference in the second optical image. The optical path difference generating member is arranged such that the optical path difference in the second optical image is symmetrical with respect to an imaging surface orthogonal to the direction of movement of the second optical image, resulting from the scan of the sample. The area control means inverts the positions of the first and second imaging areas in an imaging surface with respect to the axis as the sample is inverted in the direction of scanning.

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. In particular, when the scan direction of the sample changes, it is necessary to adjust the optical surface before and after the change of the scan direction, and the adjustment work may be complicated. 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 device and a focus method thereof that can acquire the shift direction of the focal position under the same conditions regardless of the scanning direction of the sample with a simple configuration. 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 acquires a second image based on the second optical image branched into the second optical path, and the second image, A focus control unit that controls the focal position of imaging by the first imaging unit based on the analysis result, and a first imaging region that acquires a partial image of the second optical image on the imaging surface of the second imaging unit And an area control means for setting the second imaging area, and an in-plane direction of the imaging surface disposed in the second optical path An optical path difference generating member that generates an optical path difference in the second optical image along the optical path difference generating member. The optical path difference generating member is configured with respect to the axis of the imaging surface orthogonal to the moving direction of the second optical image associated with scanning of the sample. The optical path difference between the two optical images is arranged to be symmetric, and the area control means determines the position of the first imaging area and the position of the second imaging area on the imaging surface as the scanning direction of the sample is reversed. It is characterized by reversing the axis.

  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. Further, in this image acquisition device, the position of the first imaging area and the position of the second imaging area on the imaging surface can be inverted with respect to the axis of the imaging surface in accordance with the inversion of the scanning direction of the sample. For this reason, the shift direction of the focal position can be acquired under the same conditions regardless of the scanning direction of the sample.

  In the imaging surface, it is preferable that a region where a light image conjugate with the first light image incident on the first imaging means is substantially coincident with the axis of the imaging surface. Thereby, even when the scanning direction of the sample is reversed, the positional relationship between the first imaging region, the second imaging region, and the region where the optical image conjugate with the first optical image is incident on the imaging surface is obtained. Can be maintained.

  Moreover, it is preferable that the axis of the imaging surface substantially coincides with the central axis of the imaging surface orthogonal to the moving direction of the second optical image accompanying the scanning of the sample. In this case, it is possible to reliably reverse the position of the first imaging region and the position of the second imaging region on the imaging surface accompanying the reversal of the scanning direction of the sample.

  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 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 the control by the focus control unit are provided. It is preferable that the objective lens is not driven during the position analysis, and the objective lens is moved in one direction with respect to the sample while the focus position analysis by the focus control unit is not executed. In this case, the analysis accuracy of the focal position can be ensured by not changing the positional relationship between the objective lens and the sample during the analysis of the focal position.

  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.

  In addition, the focus method of the image acquisition apparatus according to the present invention branches the light source that irradiates light toward the sample, and the optical image of the sample into the first optical path for image acquisition and the second optical path for focus control. A light guiding optical system including a light branching unit; a first imaging unit that obtains a first image based on the first light image branched into the first optical path; and a second optical unit branched into the second optical path. A second imaging means for acquiring a second image by an optical image; a focus control means for analyzing the second image and controlling a focal position of imaging by the first imaging means based on the analysis result; In the focusing method of the image acquisition apparatus, a first imaging region and a second imaging region for acquiring a partial image of the second optical image are set on the imaging surface of the second imaging unit, and the imaging surface An optical path difference generating member that generates an optical path difference in the second optical image along the in-plane direction of the 2 is arranged in the second optical path so that the optical path difference of the second optical image is symmetric with respect to the axis of the imaging plane orthogonal to the moving direction of the optical image. The position of the first imaging region and the position of the second imaging region on the imaging surface are inverted with respect to the axis of the imaging surface.

  In the focusing method of the image acquisition device, the optical path difference generating member is arranged 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 area and the position of the second imaging area on the imaging surface are reversed about the axis in accordance with the reversal of the scanning direction of the sample. For this reason, the shift direction of the focal position can be acquired under the same conditions regardless of the scanning direction of the sample.

  In addition, it is preferable that a region where a light image conjugate with the first light image incident on the first image pickup unit is substantially coincided with the axis of the image pickup surface on the image pickup surface. Thereby, even when the scanning direction of the sample is reversed, the positional relationship between the first imaging region, the second imaging region, and the region where the optical image conjugate with the first optical image is incident on the imaging surface is obtained. Can be maintained.

  It is preferable that the axis of the imaging surface is substantially coincident with the central axis of the imaging surface that is orthogonal to the moving direction of the second optical image accompanying the scanning of the sample. In this case, it is possible to reliably reverse the position of the first imaging region and the position of the second imaging region on the imaging surface accompanying the reversal of the scanning direction of the sample.

  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 the control by the focus control unit. It is preferable that the objective lens is not driven during the 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 is not executed by the focus control means. By not changing the positional relationship between the objective lens and the sample during the analysis of the focal position, the analysis accuracy of the focal position can be ensured.

  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 shift direction of the focal position can be acquired according to the scanning direction of the sample with a simple configuration.

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 (forward direction) of an optical path difference production | generation member and a 2nd imaging device. It is a figure which shows an 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. 13) 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. In macro image acquisition, the entire image of the sample S may be acquired by one imaging, or the sample S may be divided into a plurality of regions and imaged. 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 is used. Used. The first imaging device 18 has a structure in which, for example, two TDI sensors having different transfer directions are combined, and the light receiving units having a plurality of light receiving rows are located on the center side of the imaging surface. The first imaging device 18 may be a line sensor.

  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.

  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 sensors are used. A line sensor may be 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 second light image is projected onto the imaging surface 20a. The second optical image moves the imaging surface 20a in the + Z direction when the sample S is scanned in the forward direction by the stage 1, and moves the imaging surface 20a in the -Z direction when scanned in the reverse direction of the sample S. Moving. 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. Further, 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.

  The optical path difference generating member 21 is a glass member that generates an optical path difference in the second optical image along the in-plane direction of the imaging surface 20a. The optical path difference generating member 21 is disposed so that the optical path difference of the second optical image is symmetric with respect to the axis P of the imaging surface 20a orthogonal to the moving direction of the second optical image accompanying the scanning of the sample S. 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. 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, and after the imaging in the second imaging area 22B is performed, the second imaging area 22B is set. Is released.

  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 changes the position of the first imaging area 22A and the position of the second imaging area 22B on the imaging surface 20a with respect to the axis P of the imaging surface 20a as the scanning direction of the sample S is reversed. Are reversed so that they are line symmetric. This setting is appropriately executed according to the shape and arrangement of the optical path difference generating member 21. For example, in the example shown in FIGS. 7 and 8, the optical path difference generation member 21A formed of a flat glass member is arranged so as to coincide with the axis P of the imaging surface 20a. A region 22C where a light image conjugate with the first light image incident on the first image pickup device 18 is located so as to coincide with the axis P of the image pickup surface 20a. In the present embodiment, the axis P of the imaging surface 20a is in a state of being coincident with the central axis of the imaging surface 20a orthogonal to the moving direction of the second optical image accompanying the scanning of the sample S.

  As shown in FIG. 7, when the sample S is scanned in the forward direction (+ Z direction), the first imaging region 22A overlaps the optical path difference generation member 21A in the upper half region of the imaging surface 20a. The second imaging area 22B is set to an area overlapping the optical path difference generating member 21A. As shown in FIG. 8, when the sample S is scanned in the reverse direction (−Z direction), in the lower half region of the imaging surface 20a, the first imaging region 22A is separated from the optical path difference generating member 21A. The second imaging region 22B is set as a region that overlaps with the optical path difference generation 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, the interval d between the first imaging region 22A and the second imaging region 22B is made wider than the width of the shadow 23, and the first imaging region 22A and the second imaging region are located at positions avoiding the shadow 23. It is preferable to set 22B.

  In the example shown in FIGS. 9 and 10, the optical path difference generating member 21B made of a prism-shaped glass member whose thickness continuously changes toward the center in the Z direction is aligned with the axis P of the imaging surface 20a. Has been placed. The change in the thickness of the optical path difference generating member 21B is symmetric with respect to the axis P of the imaging surface, and is switched from increasing to decreasing along the Z direction. A region 22C where a light image conjugate with the first light 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. In this embodiment as well, the axis P of the imaging surface 20a coincides with the central axis of the imaging surface 20a orthogonal to the moving direction of the second optical image accompanying the scanning of the sample S.

  As shown in FIG. 9, when the sample S is scanned in the forward direction (+ Z direction), in the upper half area of the imaging surface 20a, the first imaging area 22A is the upper side, and the second imaging area 22B. Is set to be on the lower side. Also, as shown in FIG. 10, when the sample S is scanned in the reverse direction (−Z direction), the first imaging region 22A is the lower side and the second imaging in the lower half region of the imaging surface 20a. The region 22B is set to be on the upper side.

  In addition, as the optical path difference generating member 21, for example, as shown in FIG. 11A, an optical path difference in which the central portion in the Z direction has a constant thickness and the thickness decreases as both ends in the Z direction go outward. The generation member 21C may be used. For example, as shown in FIG. 11 (b), the optical path difference generation member 21D made of two glass members having a prism shape with a right-angled cross section is formed at both ends of the imaging surface 20a in the Z direction You may arrange | position so that thickness may decrease toward the center side from the side.

  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. 12 is a diagram illustrating the 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. Thus, the analysis accuracy of the focal position can be ensured by not changing the positional relationship between the objective lens 15 and the sample S during the analysis of the focal position.

  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. For example, as shown in FIG. 13, the scanning direction of the stage 1 is the scanning of the next divided region 40 in the opposite direction by moving the stage 1 in the direction orthogonal to the scanning direction after the scanning of one divided region 40 is completed. Bi-directional scanning.

  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. 14, 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. 15 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 performed 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. A waiting time W until the start is set. 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 for each divided region 40 of the sample S are acquired 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 direction 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 of the second imaging device 20 are provided by arranging the optical path difference generation member 21 in the second optical path L2. In the region 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, since the optical path length difference can be formed without branching the light in the second optical path L2 for focus control, it is possible to obtain the information on the focal position from the second optical path L2. The amount of light is suppressed, and the amount of light when the first imaging device 18 performs imaging can be secured. Further, in this image acquisition device M, the position of the first imaging region 22A and the position of the second imaging region 22B on the imaging surface 20a are line-symmetric with respect to the axis P as the scanning direction of the sample S is reversed. It is reversed so that For this reason, regardless of the scanning direction of the sample S, the shift direction of the focal position can be acquired under the same conditions.

  Further, in this image acquisition device M, from the imaging in the first imaging area 22A based on the scanning speed v of the stage and the interval d between the first imaging area 22A and the second imaging area 22B. A waiting time W until imaging in the second imaging area 22B is set. Therefore, since the light from the same position of the sample S enters the first imaging region 22A and the second imaging region 22B, the focal position of the objective lens 15 can be controlled with high accuracy.

  As the optical path difference generating member of the present embodiment, an optical path difference generating member 21 (21B to 21D) 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. 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 area 22A and the position of the second imaging area 22B by the area control unit 35. 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.

  Moreover, when using the optical path difference generation member 21 (21A) which consists of a flat glass member as an optical path difference generation member of this embodiment, the structure of the optical path difference generation member 21 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.

  Further, in the above-described embodiment, the imaging surface 20a is positioned on the near side in the scanning direction of the sample S with respect to the region 22C where the optical image conjugate with the first optical image incident on the first imaging device 18 is incident. In this way, the position of the first imaging area 22A and the position of the second imaging area 22B are set. Thereby, it is possible to acquire in advance the shift direction of the focal position with respect to the imaging position of the first imaging device 18. In addition, this invention is not restricted to the said aspect, The aspect which analyzes the shift | offset | difference direction of a focus position in the imaging position of the 1st imaging device 18 may be sufficient.

  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 21D) ... optical path difference generating member, 22A ... first imaging region, 22B ... second imaging region, 34 ... focus control unit (focus) Control means), 35 ... area control section (area control means), 36 ... objective lens control section (objective lens control means), E ... edge portion, L1 ... first optical path, L2 ... second optical path, M ... image Acquisition device, M1 ... macro image acquisition device, M2 ... micro image acquisition device, P ... axis, S ... sample.

Claims (14)

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 for acquiring a second image by a 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 optical path difference generating member is arranged such that the optical path difference of the second optical image is symmetric with respect to the axis of the imaging surface orthogonal to the moving direction of the second optical image accompanying the scanning of the sample,
The area control means reverses the position of the first imaging area and the position of the second imaging area on the imaging surface with respect to the axis in accordance with the reversal of the scanning direction of the sample. Acquisition device.   2. The region on the imaging surface where a light image conjugate with the first optical image incident on the first imaging means is substantially coincident with the axis of the imaging surface. The image acquisition device described.   3. The image according to claim 1, wherein an axis of the imaging surface substantially coincides with a central axis of the imaging surface that is orthogonal to a moving direction of the second optical image associated with scanning of the sample. Acquisition device. 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 device according to any one of claims 1 to 3, wherein a second imaging region 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 said area control means sets the said 1st imaging area and the said 2nd imaging area so that it may overlap with the part from which the thickness of the said optical path difference production | generation member differs in any one of Claims 1-4 characterized by the above-mentioned. The image acquisition device according to one item. 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 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 device according to claim 1, wherein a waiting time until imaging in the imaging region is set. 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 for acquiring a second image by a 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 a result of the analysis, and a focus method of the image acquisition apparatus,
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 generates an optical path difference in the second optical image along an in-plane direction of the imaging surface is provided on the imaging surface orthogonal to the moving direction of the second optical image associated with scanning of the sample. Arranged in the second optical path so that the optical path difference of the second optical image is symmetric about the axis;
An image characterized in that the region control means reverses the position of the first imaging region and the position of the second imaging region on the imaging surface with respect to the axis in accordance with the reversal of the scanning direction of the sample. Focus method of the acquisition device.   9. The region of the imaging surface where a light image conjugate with the first optical image incident on the first imaging means is substantially coincided with the axis of the imaging surface. Focus method of image acquisition device.   10. The image acquisition apparatus according to claim 8, wherein an axis of the imaging surface is made to substantially coincide with a central axis of the imaging surface that is orthogonal to a moving direction of the second optical image accompanying the scanning of the sample. Focus method. 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 11. The focus method for an image acquisition apparatus according to claim 8, 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 said area | region control means sets the said 1st imaging area and the said 2nd imaging area so that it may overlap with the part from which the thickness of the said optical path difference production | generation member differs, The any one of Claims 8-11 characterized by the above-mentioned. The focus method of the image acquisition device according to one item. 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 8, wherein the focus is moved in one direction.   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 8, wherein a waiting time until imaging in the imaging area is set.

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