US20200033564A1

US20200033564A1 - Image capturing apparatus and focusing method thereof

Info

Publication number: US20200033564A1
Application number: US16/592,192
Authority: US
Inventors: Hideshi Oishi
Original assignee: Hamamatsu Photonics KK
Current assignee: Hamamatsu Photonics KK
Priority date: 2011-12-19
Filing date: 2019-10-03
Publication date: 2020-01-30
Also published as: WO2014112084A1; EP3333608B1; US10571664B2; EP3333608A1; EP2947488B1; CN104937469B; DK3333608T3; EP2947488A4; US20180196237A1; CN104937469A; US20150109512A1; US9921392B2; EP2947488A1; US20170285306A9; HUE054657T2

Abstract

In the image capturing apparatus, the optical path difference producing member is disposed on the second optical path. Thereby, it is possible to suppress the amount of light when an optical image which is focused at the front of an optical image made incident into the first imaging device (front focus) and an optical image which is focused at the rear thereof (rear focus) are respectively imaged at the second imaging device and also to secure the amount of light on image pickup by the first imaging device. Further, in the image capturing apparatus, a position of the first imaging region and a position of the second imaging region on the imaging area are reversed with respect to the axis P in association with reversal of a scanning direction of the sample. Therefore, despite the scanning direction of the sample, it is possible to obtain a deviation direction of the focus position under the same conditions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/889,332, filed Feb. 6, 2018, which is a continuation of U.S. patent application Ser. No. 14/398,011, now U.S. Pat. No. 9,921,32, filed Oct. 30, 2014. U.S. patent application Ser. No. 14/398,011 is a § 371 national stage application of PCT/JP2013/050853, filed on Jan. 17, 2013, which claims priority to Japanese Patent Application No. 2011-277539, filed Dec. 19, 2011. The entire contents of each of the aforementioned applications are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an image capturing apparatus which is used for capturing images of a sample, etc., and also relates to a focusing method thereof.

BACKGROUND ART

Image capturing apparatuses include a virtual microscope apparatus in which, for example, an imaging region of a sample is in advance divided into a plurality of regions to image the divided regions at a high magnification and, thereafter, to synthesize the regions. In capturing images by using the virtual microscope as described above, conventionally, as conditions for picking up images of a sample such as a biological sample, a focus map which covers an entire region of the sample is set to capture images of the sample, while focus control is performed based on the focus map.
In preparation of the focus map, at first, an image capturing apparatus equipped with a macro optical system is used to capture an entire sample as a macro image. Next, the thus captured macro image is used to set an image pickup range of the sample and also the range is divided into a plurality of regions to set a focus obtaining position for each of the divided regions. After the focus obtaining position has been set, the sample is transferred to the image capturing apparatus equipped with a micro optical system to obtain a focus position at the thus set focus obtaining position, thereby preparing a focus map with reference to the focus position.
However, in preparation of the above-described focus maps, there has been a problem that processing needs time. Further, suppression of intervals and the number of focuses to be obtained would reduce the time necessary for the processing. In this case, however, there has been a problem of reduction in focus accuracy. Therefore, development of dynamic focus for capturing images of a sample at a high magnification, with a focus position being obtained, is now underway. The dynamic focus is a method in which a present direction of the focus position deviating from the height of an objective lens is detected based on a difference in light intensity or a difference in contrast between an optical image which is focused at the front of an optical image made incident into an imaging device for capturing an image (front focus) and an optical image which is focused at the rear thereof (rear focus), thereby allowing the objective lens to move in a direction at which the deviation is cancelled to capture an image.
A microscope system disclosed, for example, in Patent Document 1, is provided with a second imaging unit which images a region at the front of a region imaged by a first imaging unit, an automatic focusing control unit which adjusts a focusing position of an objective lens at an imaging position of the first imaging unit based on an image picked up by the second imaging unit, and a timing control unit which synchronizes timing at which a divided region moves from an imaging position of the second imaging unit to the imaging position of the first imaging unit with timing at which an image forming position of the divided region imaged by the second imaging unit is positioned at an imaging area of the first imaging unit depending on a distance between the divided regions and a speed at which a sample moves. Further, in a microscope apparatus disclosed, for example, in Patent Document 2 or Patent Document 3, a glass member is used to make a difference in optical path length inside a light guiding optical system for focus control.

CITATION LIST

Patent Literature

[Patent Document 1] Japanese Patent Application Laid-Open No. 2011-081211

[Patent Document 2] Japanese Patent Publication No. WO2005/114287
[Patent Document 3] Japanese Patent Publication No. WO2005/114293

SUMMARY OF INVENTION

Technical Problem

In the microscope system described in Patent Document 1, a half mirror and a mirror are used to form an optical path difference optical system, by which light different in optical path length is made incident into each of two imaging regions of the second imaging unit. In the conventional microscope system, for example, a line sensor is used to constitute a first imaging unit and a second imaging unit. In the line sensor, it is important to secure an amount of light for capturing a clear image due to short exposure time. However, in the conventional microscope system, light is divided by the optical path difference optical system. Thus, there is posed such a problem that it is difficult to secure an amount of light.

Technical Problem

Further, in the conventional microscope system, an optical surface of an optical path dividing unit is inclined, by which a region imaged by the second imaging unit is adjusted so as to be on the front side of the sample in a scanning direction with respect to a region imaged by the first imaging unit, thereby capturing in advance a direction at which the focus position will deviate. However, the above-described configuration has difficulty in adjusting the optical surface, which poses a problem. In particular, where the sample changes in scanning direction, it is necessary to adjust the optical surface before or after the change in scanning direction, which may result in complicated adjustment work. Further, the half mirror and the mirror are used to constitute the optical path difference optical system. Thus, there has also been a problem that light which has passed through the optical path difference optical system is less likely to gather on an imaging area of the second imaging unit.
The present invention has been made in order to solve the above-described problems, an object of which is to provide an image capturing apparatus which is simple in configuration and capable of obtaining a direction at which a focus position deviates under the same conditions, despite a scanning direction of a sample, and also to provide a focusing method thereof.

Solution to Problem

In order to solve the above-described problems, the image capturing apparatus of the present invention is characterized by having a stage on which a sample is placed, a stage control unit which scans the stage at a predetermined speed, a light source which radiates light to the sample, a light guiding optical system including a light dividing unit which divides an optical image of the sample into a first optical path for capturing an image and a second optical path for focus control, a first imaging unit which captures a first image by a first optical image divided into the first optical path, a second imaging unit which captures a second image by a second optical image divided into the second optical path, a focus control unit which analyzes the second image to control a focus position of the image pickup by the first imaging unit based on the analysis result, a region control unit which sets a first imaging region and a second imaging region for capturing a partial image of the second optical image on an imaging area of the second imaging unit, and an optical path difference producing member which is disposed on the second optical path to give an optical path difference to the second optical image along an in-plane direction of the imaging area, in which the optical path difference producing member is disposed in such a manner that an optical path difference of the second optical image is symmetrical with respect to an axis of the imaging area orthogonal to a direction at which the second optical image moves in association with scanning of the sample, and the region control unit reverses a position of the first imaging region and a position of the second imaging region on the imaging area with respect to the axis in association with reversal of the scanning direction of the sample.
In the image capturing apparatus, the optical path difference producing member is disposed on the second optical path. Thereby, at the first imaging region and the second imaging region of the second imaging unit, it is possible to image respectively an optical image which is focused at the front of an optical image made incident into the first imaging unit (front focus) and an optical image which is focused at the rear thereof (rear focus). In the image capturing apparatus, it is possible to make a difference in optical path length without dividing light on the second optical path for focus control. Therefore, an amount of light at the second optical path necessary for obtaining information on a focus position can be suppressed to secure an amount of light on the image pickup by the first imaging unit. Further, in the image capturing apparatus, it is possible to reverse the position of the first imaging region and the position of the second imaging region on the imaging area with respect to the axis of the imaging area in association with reversal of the scanning direction of the sample. Therefore, despite the scanning direction of the sample, it is possible to obtain a direction at which the focus position deviates under the same conditions.
It is also preferable that a region into which an optical image is made incident which is conjugate to the first optical image made incident into the first imaging unit is substantially in alignment with the axis of the imaging area on the imaging area. Thereby, even where the sample is reversed in scanning direction, it is possible to keep a positional relationship between the first imaging region, the second imaging region and the region into which an optical image is made incident which is conjugate to the first optical image on the imaging area.
It is also preferable that the axis of the imaging area is substantially in alignment with a central axis of the imaging area orthogonal to a direction at which the second optical image moves in association with 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 area in association with reversal of the scanning direction of the sample.
Still further, it is preferable that the optical path difference producing member is a flat plate member which is disposed so as to overlap at least on a part of the imaging area and that the region control unit sets the first imaging region and the second imaging region respectively to give a region which will overlap on the flat plate member and a region which will not overlap on the flat plate member in order to avoid a shadow of the second optical image by an edge part of the flat plate member. In this case, use of the flat plate member enables the optical path difference producing member to be simple in configuration. Further, the edge part of the flat plate member forms the shadow of the second optical image at the imaging area of the second imaging device. Therefore, the first imaging region and the second imaging region are set so as to avoid the shadow, thus making it possible to secure accurate control of the focus position.
It is also preferable that the optical path difference producing member is a member having a part which undergoes a continuous change in thickness along an in-plane direction of the imaging area and that the region control unit sets the first imaging region and the second imaging region so as to overlap on the part of the optical path difference producing member which is different in thickness. In this case, adjustment of a position of the first imaging region and that of the second imaging region makes it possible to adjust freely an interval between the front focus and the rear focus. Thereby, it is possible to detect a focus position of the sample at high accuracy.
It is also preferable that there are provided an objective lens which faces to a sample and an objective lens control unit which controls a position of the objective lens relatively with respect to the sample based on control by the focus control unit, in which the objective lens control unit will not actuate the objective lens during analysis of the focus position which is being performed by the focus control unit and will allow the objective lens to move with respect to the sample in one direction during analysis of the focus position which is not being performed by the focus control unit. In this case, since no change in positional relationship will take place between the objective lens and the sample during analysis of the focus position, it is possible to secure analysis accuracy of the focus position.
It is also preferable that the region control unit sets waiting time from image pickup at the first imaging region to image pickup at the second imaging region based on a scanning speed of the stage and an interval between the first imaging region and the second imaging region. Therefore, since light from the same position of the sample is made incident into the first imaging region and the second imaging region, it is possible to control a focus position at high accuracy.
Further, the focusing method of the image capturing apparatus in the present invention is a focusing method of an image capturing apparatus which is characterized by having a light source which radiates light to a sample, a light conductive optical system including a light dividing unit which divides an optical image of the sample into a first optical path for capturing an image and a second optical path for focus control, a first imaging unit which captures a first image by the first optical image divided into the first optical path, a second imaging unit which captures a second image by the second optical image divided into the second optical path, and a focus control unit which analyzes the second image to control a focus position of the image by the first imaging unit based on an analysis result thereof, in which the first imaging region and the second imaging region for capturing a partial image of the second optical image are set on the imaging area of the second imaging unit, an optical path difference producing member which gives an optical path difference to the second optical image along an in-plane direction of the imaging area is disposed on the second optical path in such a manner that the optical path difference of the second optical image is symmetrical with respect to the axis of the imaging area orthogonal to a direction at which the second optical image moves in association with scanning of the sample, and the region control unit reverses a position of the first imaging region and a position of the second imaging region on the imaging area with respect to the axis of the imaging area in association with reversal of the scanning direction of the sample.
In the focusing method of the image capturing apparatus, the optical path difference producing member is disposed on the second optical path. Thereby, at the first imaging region and the second imaging region of the second imaging unit, it is possible to image respectively an optical image which is focused at the front of an optical image made incident into the first imaging unit (front focus) and an optical image which is focused at the rear thereof (rear focus). In the focusing method, it is possible to make a difference in optical path length without dividing light on the second optical path for focus control. Therefore, the amount of light at the second optical path necessary for obtaining information on a focus position can be suppressed to secure the amount of light on image pickup by the first imaging unit. Further, in the focusing method, the position of the first imaging region and the position of the second imaging region on the imaging area are reversed with respect to the axis in association with reversal of the scanning direction of the sample. Therefore, despite the scanning direction of the sample, it is possible to obtain a deviation direction of the focus position under the same conditions.
It is also preferable that a region into which an optical image is made incident which is conjugate to the first optical image made incident into the first imaging unit on the imaging area is made substantially in alignment with the axis of the imaging area. Thereby, even where the sample is reversed in scanning direction, it is possible to keep a positional relationship between the first imaging region, the second imaging region and the region into which an optical image is made incident which is conjugate to the first optical image on the imaging area.
It is preferable that the axis of the imaging area is made substantially in alignment with the central axis of the imaging area orthogonal to a direction at which the second optical image moves in association with 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 area in association with reversal of the scanning direction of the sample.
It is also preferable that as the optical path difference producing member, there is used a flat plate member which is disposed so as to overlap at least on a part of the imaging area and in order to avoid a shadow of the second optical image by an edge part of the flat plate member, the first imaging region and the second imaging region are set by the region control unit respectively so as to give a region which will overlap on the flat plate member and a region which will not overlap on the flat plate member. In this case, use of the flat plate member enables the optical path difference producing member to be made simple in configuration. Further, the edge part of the flat plate member forms the shadow of the second optical image at an imaging area of the second imaging device. Therefore, the first imaging region and the second imaging region are set so as to avoid the shadow, thus making it possible to secure accurate control of the focus position.
It is also preferable that as the optical path difference producing member, there is used a member which has a part undergoing a continuous change in thickness along an in-plane direction of the imaging area and that the first imaging region and the second imaging region are set by the region control unit so as to overlap on the part of the optical path difference producing member which is different in thickness. In this case, adjustment of a position of the first imaging region and that of the second imaging region makes it possible to freely adjust an interval between the front focus and the rear focus. It is, thereby, possible to detect a focus position of the sample at high accuracy.
It is also preferable that the image capturing apparatus is provided with an objective lens which faces to a sample and an objective lens control unit which controls a position of the objective lens relatively with respect to the sample based on control by the focus control unit, in which the objective lens control unit will not drive the objective lens during analysis of the focus position which is being performed by the focus control unit and will allow the objective lens to move with respect to the sample in one direction during analysis of the focus position which is not being performed by the focus control unit. Since no change in positional relationship will take place between the objective lens and the sample during analysis of the focus position, it is possible to secure analysis accuracy of the focus position.
It is also preferable that waiting time from image pickup at the first imaging region to image pickup at the second imaging region is set by the region control unit based on a scanning speed of the stage and an interval between the first imaging region and the second imaging region. Therefore, since light from the same position of the sample is made incident into the first imaging region and the second imaging region, it is possible to control the focus position at high accuracy.

Advantageous Effects of Invention

The present invention is able to obtain a direction at which the focus position deviates by a simple configuration according to the scanning direction of the sample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing which shows one embodiment of a macro image capturing device which constitutes an image capturing apparatus of the present invention.

FIG. 2 is a drawing which shows one embodiment of a micro image capturing device which constitutes the image capturing apparatus of the present invention.

FIG. 3 is a block diagram which shows functional components of the image capturing apparatus.

FIG. 4A and FIG. 4B are drawings which show an analysis result of a contrast value where a distance to the surface of a sample is in agreement with the focal length of an objective lens.

FIG. 5A and FIG. 5B are drawings which show an analysis result of a contrast value where a distance to the surface of the sample is longer than the focal length of the objective lens.

FIG. 6A and FIG. 6B are drawings which show an analysis result of a contrast value where a distance to the surface of the sample is shorter than the focal length of the objective lens.

FIG. 7A and FIG. 7B are drawings which show one example of an optical path difference producing member and a second imaging device (forward direction).

FIG. 8A and FIG. 8B are drawings which show one example of the optical path difference producing member and the second imaging device (reverse direction).

FIG. 9A and FIG. 9B are drawings which show another example of the optical path difference producing member and the second imaging device (forward direction).

FIG. 10A and FIG. 10B are drawings which show another example of the optical path difference producing member and the second imaging device (reverse direction).

FIG. 11A and FIG. 11B are drawings which show a modified example of the optical path difference producing member.

FIG. 12 is a drawing which shows a relationship of the distance between the objective lens and the surface of the sample with respect to scanning time of a stage.

FIG. 13 is a drawing which shows control of a scanning direction of the stage by a stage control portion.

FIG. 14A and FIG. 14B are drawings which show control of a scanning speed of the stage by the stage control portion.

FIG. 15 is a flow chart which shows motions of the image capturing apparatus.

DESCRIPTION OF EMBODIMENTS

Hereinafter, a description will be given in detail of preferred embodiments of the image capturing apparatus and the focusing method of the image capturing apparatus in the present invention with reference to drawings.
FIG. 1 is a drawing which shows one embodiment of the macro image capturing device which constitutes the image capturing apparatus of the present invention. FIG. 2 is a drawing which shows one embodiment of the micro image capturing device which constitutes the image capturing apparatus of the present invention. As shown in FIG. 1 and FIG. 2, an image capturing apparatus M is constituted with a macro image capturing device M1 for capturing a macro image of a sample S and a micro image capturing device M2 for capturing a micro image of the sample S. The image capturing apparatus M is an apparatus which sets, for example, a plurality of line-shaped divided regions 40 with respect to the macro image captured by the macro image capturing device M1 (refer to FIG. 13) and produces a virtual micro image by capturing and synthesizing each of the divided regions 40 by the micro image capturing device M2 at a high magnification.
As shown in FIG. 1, the macro image capturing device M1 is provided with a stage 1 which supports the sample S. The stage 1 is an XY stage which is actuated in a horizontal direction by a motor or an actuator such as a stepping motor (pulse motor) or a piezo actuator, for example. The sample S which is observed by using the image capturing apparatus M is, for example, a biological sample such as cells and placed on the stage 1 in a state of being sealed on a slide glass. The stage 1 is actuated inside the XY plane, by which an imaging position with respect to the sample S is allowed to move.
The stage 1 is able to move back and forth between the macro image capturing device M1 and the micro image capturing device M2 and provided with functions to deliver the sample S between the devices. It is acceptable that when a macro image is captured, an entire image of the sample S is picked up at one time or the sample S is divided into a plurality of regions to pick up each of the images. It is also acceptable that the stage 1 is installed both on the macro image capturing device M1 and on the micro image capturing device M2.
A light source 2 which radiates light to the sample S and a condensing lens 3 which concentrates light from the light source 2 at the sample S are disposed on a bottom of the stage 1. It is acceptable that the light source 2 is disposed so as to radiate light obliquely to the sample S. Further, a light guiding optical system 14 which guides an optical image from the sample S and an imaging device 5 which images the optical image of the sample S are disposed on an upper face of the stage 1. The light guiding optical system 14 is provided with an image forming lens 6 which forms the optical image from the sample S at an imaging area of the imaging device 5. Still further, the imaging device 5 is an area sensor which is capable of capturing, for example, a two-dimensional image. The imaging device 5 captures an entire image of the optical image of the sample S made incident into the imaging area via the light guiding optical system 14 and is housed at a virtual micro image housing portion 39 to be described later.
As shown in FIG. 2, the micro image capturing device M2 is provided on the bottom of the stage 1 with a light source 12 and a condensing lens 13, as with the macro image capturing device M1. Further, a light guiding optical system 14 which guides an optical image from the sample S is disposed on the upper face of the stage 1. The optical system which radiates light from the light source 12 to the samples may include an excitation light radiating optical system which radiates excitation light to the sample S and a dark-field illuminating optical system which captures a dark-field image of the sample S.
The light guiding optical system 14 is provided with an objective lens 15 disposed so as to face to the sample S and a beam splitter (light dividing unit) 16 disposed at a rear stage of the objective lens 15. The objective lens 15 is provided with a motor and an actuator such as a stepping motor (pulse motor) and a piezo actuator for actuating the objective lens 15 in a Z direction orthogonal to a face on which the stage 1 is placed. A position of the objective lens 15 in the Z direction is changed by these actuation units, thus making it possible to adjust a focus position of image pickup when an image of the sample S is captured. It is acceptable that the focus position is adjusted by changing a position of the stage 1 in the Z direction or by changing positions of both the objective lens 15 and the stage 1 in the Z direction.
The beam splitter 16 is a portion which divides an optical image of the sample S into a first optical path L1 for capturing an image and a second optical path L2 for focus control. The beam splitter 16 is disposed at an angle of approximately 45 degrees with respect to an optical axis from the light source 12. In FIG. 2, an optical path passing through the beam splitter 16 is given as the first optical path L1, while an optical path reflected at the beam splitter 16 is given as the second optical path.
On the first optical path L1, there are disposed an image forming lens 17 which forms an optical image of the sample S (first optical image) which has passed through the beam splitter 16 and a first imaging device (first imaging unit) 18 in which an imaging area is disposed at an image forming position of the image forming lens 17. The first imaging device 18 is a device which is capable of capturing a one-dimensional image (first image) by the first optical image of the sample S, including, for example, a two-dimensional CCD sensor which is capable of realizing, TDI (time delay integration) actuation. The first imaging device 18 has a structure formed by combining, for example, two TDI sensors different in transfer direction and in which light receiving portions, each of which has plural stages of light receiving arrays, are positioned at the center of the imaging area. It is noted that the first imaging device 18 may be a line sensor.
Further, in a method which captures sequentially images of the sample S, with the stage 1 controlled at a constant speed, the first imaging device 18 may be a device such as a CMOS sensor or a CCD sensor which is capable of capturing a two-dimensional image. First images picked up by the first imaging device 18 are sequentially stored at a temporary storage memory such as a lane buffer, thereafter, compressed and output at an image producing portion 38 to be described later.
On the other hand, on the second optical path L2, there are disposed a view-field adjusting lens 19 which contracts an optical image of a sample reflected by the beam splitter 16 (second optical image) and a second imaging device (second imaging unit) 20. Further, at a front stage of the second imaging device 20, there is disposed an optical path difference producing member 21 which gives an optical path difference to the second optical image. It is preferable that the view-field adjusting lens 19 is constituted in such a manner that the second optical image is formed at the second imaging device 20 in a dimension similar to that of the first optical image.
The second imaging device 20 is a device which is capable of capturing a two-dimensional image (second image) by the second optical image of the sample S, including, for example, sensors such as a CMOS (complementary metal oxide semiconductor) and a CCD (charge coupled device). It is also acceptable that a line sensor is used.
The imaging area 20 a of the second imaging device 20 is disposed so as to be substantially in alignment with an XZ plane orthogonal to the second optical path L2. The second optical image is projected on the imaging area 20 a. The second optical image is such that where the sample S is scanned by the stage 1 in the forward direction, the imaging area 20 a will move in a +Z direction and where the sample S is scanned in the reverse direction, the imaging area 20 a will move in a −Z direction. The first imaging region 22A and the second imaging region 22B, each of which captures a partial image of the second optical image, are set on the imaging area 20 a (for example, refer to FIG. 7A and FIG. 7B). The first imaging region 22A and the second imaging region 22B are set in a direction perpendicular to a direction (scanning direction: Z direction) at which the second optical image moves on the imaging area 20 a in association with scanning of the sample S. Further, the first imaging region 22A and the second imaging region 22B are set, with a predetermined interval kept, both of which captures a part of the second optical image in a line shape. Thereby, an optical image which is at the same region as that of the first optical image of the sample S captured by the first imaging device 18 can be captured as the second optical image at the first imaging region 22A and the second imaging region 22B.
Thereby, the second imaging device 20 is able to capture an optical image which is focused at the front of a first optical image made incident into the first imaging device 18 (front focus) and an optical image which is focused at the rear thereof (rear focus) based on the position of the first imaging region 22A and that of the second imaging region 22B. A focus difference between the front focus and the rear focus is dependent on a difference between a thickness and an index of refraction of the optical path difference producing member 21 through which the second optical image made incident into the first imaging region 22A passes and a thickness and an index of refraction of the optical path difference producing member 21 through which the second optical image made incident into the second imaging region 22B passes.
The optical path difference producing member 21 is a glass member which gives an optical path difference to the second optical image along an in-plane direction of the imaging area 20 a. The optical path difference producing member 21 is disposed in such a manner that an optical path difference of the second optical image is symmetrical with respect to the axis P of the imaging area 20 a orthogonal to a direction at which the second optical image moves in association with scanning of the sample S. Further, it is preferable that the optical path difference producing member 21 is disposed in such a manner that a face which faces to the second imaging device 20 is parallel with the imaging area (light receiving face) 20 a of the second imaging device. Thereby, it is possible to reduce deflection of light by the face which faces to the second imaging device 20 and also to secure the amount of light which is received by the second imaging device 20. There will be described later a shape of the optical path difference producing member 21 and an example in which the first imaging region 22A and the second imaging region 22B are disposed on the imaging area 20 a.
FIG. 3 is a block diagram which shows functional components of the image capturing apparatus. As shown in the diagram, the image capturing apparatus M is provided with a computer system having housing portions such as a CPU, a memory, a communication interface and a hard disk, an operation portion 31 such as a keyboard, a monitor 32 etc. The functional components of the control portion 33 include a focus control portion 34, a region control portion 35, an objective lens control portion 36, a stage control portion 37, an image producing portion 38 and a virtual micro image housing portion 39.
The focus control portion 34 is a portion which analyzes a second image captured by the second imaging device 20 to control a focus position of an image picked up by the first imaging device 18 based on the analysis result. More specifically, the focus control portion 34 first determines a difference between a contrast value of the image captured at the first imaging region 22A and a contrast value captured at the second imaging region 22B in the second imaging device 20.
Here, as shown in FIG. 4A and FIG. 4B, where a focus position of the objective lens 15 is in alignment with the surface of the sample S, an image contrast value of the front focus captured at the first imaging region 22A is substantially in agreement with an image contrast value of the rear focus captured at the second imaging region 22B. Thereby, a difference value between them is almost zero.
On the other hand, as shown in FIG. 5A and FIG. 5B, where a distance to the surface of the sample S is longer than a focal length of the objective lens 15, an image contrast value of the rear focus captured at the second imaging region 22B is greater than an image contrast value of the front focus captured at the first imaging region 22A. Therefore, a difference value between them is a positive value. In this case, the focus control portion 34 outputs instruction information to the objective lens control portion 36 so as to be actuated in a direction at which the objective lens 15 is brought closer to the sample S.
Further, as shown in FIG. 6A and FIG. 6B, where a distance to the surface of the samples is shorter than a focal length of the objective lens 15, an image contrast value of the rear focus captured at the second imaging region 22B is smaller than an image contrast value of the front focus captured at the first imaging region 22A. Therefore, a difference value between them is a negative value. In this case, the focus control portion 34 outputs instruction information to the objective lens control portion 36 so as to be actuated in a direction at which the objective lens 15 is brought away from the sample S.
The region control portion 35 is a portion which controls a position of the first imaging region 22A and a position of the second imaging region 22B at the imaging area 20 a of the second imaging device 20. The region control portion 35 sets at first the first imaging region 22A at a predetermined position based on operation from the operation portion 31 and releases the setting of the first imaging region 22A after image pickup at the first imaging region 22A. Then, the region control portion 35 sets the second imaging region 22B, with a predetermined interval kept in the Z direction from the first imaging region 22A, and releases the setting of the second imaging region 22B after image pickup at the second imaging region 22B.
At this time, waiting time W from image pickup at the first imaging region 22A to image pickup at the second imaging region 22B is set based on an interval d between the first imaging region 22A and the second imaging region 22B and a scanning velocity v of the stage 1. For example, where the waiting time W is given as time W1 from the start of image pickup at the first imaging region 22A to the start of image pickup at the second imaging region 22B, it is possible to determine the waiting time with reference to a formula of W1=d/v−e1−st, with consideration given to exposure time e1 of image pickup at the first imaging region 22A and time st from release of the setting of the first imaging region 22A to the setting of the second imaging region 22B.
Further, where waiting time W is given as waiting time W2 from completion of image pickup at the first imaging region 22A to start of image pickup at the second imaging region 22B, it is possible to determine the waiting time with reference to a formula of W2=d/v−st, with consideration given to time st from release of setting of the first imaging region 22A to setting of the second imaging region 22B. Still further, an interval d between the first imaging region 22A and the second imaging region 22B is set based on a difference in optical path length given by the optical path difference producing member 21. However, the interval d actually corresponds to a distance of the sample S on a slide glass. Eventually, it is necessary to convert the interval d to the number of pixels of the second imaging region 22B. Where a pixel size of the second imaging device 20 is expressed in terms of AFpsz and magnification is expressed in terms of AFmag, the number of pixels dpix corresponding to the interval d can be determined with reference to a formula of dpix=d÷(AFpsz/AFmag).
Further, the region control portion 35 reverses the position of the first imaging region 22A and the position of the second imaging region 22B on the imaging area 20 a so as to give line symmetry to the axis P of the imaging area 20 a in association of reversal of the scanning direction of the sample S. This setting is carried out, whenever necessary, depending on the shape and arrangement of the optical path difference producing member 21. For example, in the examples shown in FIG. 7A and FIG. 7B, and FIG. 8A and FIG. 8B, the optical path difference producing member 21A which is formed with a flat plate-shaped glass member is disposed so as to be in alignment with the axis P of the imaging area 20 a. A region 22C into which an optical image is made incident which is conjugate to the first optical image made incident into the first imaging device 18 is positioned so as to be in alignment with the axis P of the imaging area 20 a. In the present embodiment, the axis P of the imaging area 20 a is kept in alignment with the central axis of the imaging area 20 a orthogonal to a direction at which the second optical image moves in association with scanning of the sample S.
Then, as shown in FIG. 7A and FIG. 7B, where the sample S is scanned in the forward direction (+Z direction), at an upper half region of the imaging area 20, the first imaging region 22A is set at a region which will not overlap on the optical path difference producing member 21A, and the second imaging region 22B is set at a region which will overlap on the optical path difference producing member 21A. Further, as shown in FIG. 8A and FIG. 8B, where the sample S is scanned in the reverse direction (−Z direction), at a lower half region of the imaging area 20 a, the first imaging region 22A is set at a region which will not overlap on the optical path difference producing member 21A, and the second imaging region 22B is set at a region which will overlap on the optical path difference producing member 21A.
There is a fear that an edge part E of the optical path difference producing member 21A may form a shadow 23 of the second optical image on the imaging area 20 a. Therefore, it is preferable that 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 that the first imaging region 22A and the second imaging region 22B are set at a position so as to avoid the shadow 23.
Further, in the examples shown in FIG. 9A and FIG. 9B, and FIG. 10A and FIG. 10B, an optical path difference producing member 21B composed of a prism-shaped glass member which undergoes a continuous change in thickness toward the center thereof in the Z direction is disposed so as to be in alignment with the axis P of the imaging area 20 a. The optical path difference producing member 21B changes in thickness so as to give a symmetric shape at the center of the axis P of the imaging area in which the thickness is switched from an increase to a decrease along the Z direction. The region 22C into which an optical image is made incident which is conjugate to the first optical image made incident into the first imaging device 18 is positioned so as to be in alignment with the center of the imaging area 20 a in the Z direction. In the present embodiment as well, the axis P of the imaging area 20 a is kept in alignment with the central axis of the imaging area 20 a orthogonal to a direction at which the second optical image moves in association with scanning of the sample S.
Moreover, as shown in FIG. 9A and FIG. 9B, where the sample S is scanned in the forward direction (+Z direction), at an upper half region of the imaging area 20 a, the first imaging region 22A and the second imaging region 22B are set respectively so as to be on the upper side and on the lower side. Further, as shown in FIG. 10A and FIG. 10B, where the sample S is scanned in the reverse direction (−Z direction), at a lower half region of the imaging area 20 a, the first imaging region 22A and the second imaging region 22B are set respectively so as to be on the lower side and on the upper side.
Moreover, it is acceptable that, as the optical path difference producing member 21, for example, as shown in FIG. 11A, there is used the optical path difference producing member 21C whose central part in the Z direction is constant in thickness and which decreases in thickness moving towards the outside the both ends thereof in the Z direction. For example, as shown in FIG. 11B, it is also acceptable that an optical path difference producing member 21D composed of two glass members, each of which has a triangular prism-shaped cross section, is disposed so as to decrease in thickness moving towards the center of the imaging area 20 a from both ends thereof in the Z direction.
Where there is set a position of the region 22C into which an optical image is made incident which is conjugate to the first optical image made incident into the first imaging device 18 on the imaging area 20 a, a distance between the stage 1 and the objective lens 15 is at first fixed to adjust a position of the stage 1 in such a manner that a cross line of a calibration slide is positioned at the center of a visual field of the first imaging device 18. Then, the second imaging device 20 is adjusted for its back focus in such a manner that the cross line of the calibration slide comes into a visual field of the second imaging device 20. Finally, an in-plane direction position of the second imaging device 20 is adjusted in such a manner that the cross line of the calibration slide is positioned at a desired site of the imaging area 20 a of the second imaging device 20.
The objective lens control portion 36 is a portion which controls actuation of the objective lens 15. Upon receiving instruction information output from the focus control portion 34, the objective lens control portion 36 actuates the objective lens 15 in the Z direction in accordance with contents of the instruction information. It is, thereby, possible to adjust a focus position of the objective lens 15 with respect to the sample S.
The objective lens control portion 36 will not actuate the objective lens 15 during analysis of the focus position which is being performed by the focus control portion 34 and will actuate the objective lens 15 only in one direction along the Z direction until start of analysis of a next focus position. FIG. 12 is a drawing which shows a relationship of the distance between the objective lens and the surface of the sample with respect to scanning time of the stage. As shown in the drawing, during scanning of the sample S, there will take place alternately an analysis period A of the focus position and an objective lens actuation period B based on an analysis result thereof. As described so far, no change in positional relationship takes place between the objective lens 15 and the sample S during analysis of the focus position, thus making it possible to secure analysis accuracy of the focus position.
The stage control portion 37 is a portion which controls actuation of the stage 1. More specifically, the stage control portion 37 allows the stage 1 on which the sample S is placed to be scanned at a predetermined speed based on operation by the operation portion 31. The stage 1 is scanned, by which an imaging field of the sample S moves relatively and sequentially at the first imaging device 18 and the second imaging device 20. Scanning of the stage 1 is, as shown in FIG. 13, for example, bi-directional scanning in which after completion of scanning of one divided region 40, the stage 1 is allowed to move in a direction orthogonal to the scanning direction and a next divided region 40 is scanned in the opposite direction.
Although the stage 1 is scanned at a constant speed while images are captured, actually, immediately after the start of scanning, there is a period during which the scanning speed is unstable due to influences of vibrations of the stage 1 etc. Thus, as shown in FIG. 14A and FIG. 14B, it is preferable that there is set a scanning width longer than the divided region 40 and an acceleration period C for accelerating the stage 1, a stabilization period D for stabilizing a scanning speed of the stage 1 and a slowing-down period F for slowing down the stage 1 are allowed to take place individually when scanning is performed outside the divided region 40. It is, thereby, possible to capture an image in synchronization with a constant speed period E during which the stage 1 is scanned at a constant speed. It is acceptable that image pickup is started during the stabilization period D and a data part obtained during the stabilization period D is deleted after the image has been captured. The above-described method is desirable when used for an imaging device which requires void reading of data.
The image producing portion 38 is a portion at which a captured image is synthesized to produce a virtual micro image. The image producing portion 38 receives sequentially first images output from the first imaging device 18, that is, images of individual divided regions 40, synthesizing these images to produce an entire image of the sample S. Then, based on the thus synthesized image, prepared is an image, the resolution of which is lower than that of the synthesized image, and housed in a virtual micro image housing portion 39 by associating a high resolution image with a low resolution image. It is acceptable that an image captured by the macro image capturing device M1 is also associated at the virtual micro image housing portion 39. It is also acceptable that the virtual micro image is housed as one image or plurally divided images.
Next, a description will be given of motions of the above-described image capturing apparatus M.
FIG. 15 is a flow chart which shows motions of the image capturing apparatus M. As shown in the flow chart, at the image capturing apparatus M, at first, a macro image of the sample S is captured by the macro image capturing device M1 (Step S01). The thus captured macro image is subjected to binarization by using, for example, a predetermined threshold value and, thereafter, displayed on a monitor 32. A scope for capturing micro images from macro images is set by automatic setting based on a predetermined program or manual setting by an operator (Step S02).
Then, the sample S is transferred to the micro image capturing device M2 and focusing conditions are set (Step S03). Here, as described above, based on a scanning velocity v of the stage 1 and an interval d between the first imaging region 22A and the second imaging region 22B, a waiting time W is set up to the start of image pickup at the second imaging region 22B. It is more preferable that consideration is given to exposure time e1 of image pickup at the first imaging region 22A, time st from release of setting of the first imaging region 22A to setting of the second imaging region 22B etc.
After the focusing conditions have been set, scanning of the stage 1 is started to capture a micro image for each of the divided regions 40 of the sample S by the micro image capturing device M2 (Step S04). In capturing the micro image by the first imaging device 18, at the second imaging device 20, a deviating direction of the objective lens 15 with respect to the sample S is analyzed based on a difference in contrast value between the front focus and the rear focus by the first imaging region 22A and the second imaging region 22B, thereby adjusting a position of the objective lens 15 in real time. After micro images have been captured completely for all the divided regions 40, the thus captured micro images are synthesized to produce a virtual micro image (Step S05).
As described so far, in the image capturing apparatus M, the optical path difference producing members 21 are disposed on the second optical path L2. Thereby, at the first imaging region 22A and the second imaging region 22B of the second imaging device 20, it is possible to image respectively an optical image which is focused at the front of an optical image made incident into the first imaging device 18 (front focus) and an optical image which is focused at the rear thereof (rear focus). In the image capturing apparatus M, a difference in optical path length can be made without dividing light on the second optical path L2 for focus control. Therefore, it is possible to suppress the amount of light at the second optical path L2 necessary for obtaining information on a focus position and to secure the amount of light on image pickup at the first imaging device 18. Further, in the image capturing apparatus M, a position of the first imaging region 22A and a position of the second imaging region 22B on the imaging area 20 a are reversed so as to give line symmetry with respect to the axis P in association with reversal of the scanning direction of the sample S. Therefore, despite the scanning direction of the sample S, it is possible to obtain a deviation direction of the focus position under the same conditions.
Further, in the image capturing apparatus M, based on a scanning velocity v of the stage and an interval d between the first imaging region 22A and the second imaging region 22B, a waiting time W is set from image pickup at the first imaging region 22A to image pickup at the second imaging region 22B. As a result, light from the same position of the sample S is made incident into the first imaging region 22A and the second imaging region 22B. Thus, it is possible to control a focus position of the objective lens 15 at high accuracy.
Where, as the optical path difference producing member of the present embodiment, there are used optical path difference producing members 21 (21B to 21D) composed of a glass member which has a part changing in thickness along an in-plane direction of the imaging area 20 a at the imaging device 20, the region control portion 35 is used to adjust a position of the first imaging region 22A and a position of the second imaging region 22B. Thereby, it is possible to freely adjust an interval between the front focus and the rear focus. Accordingly, for example, where a plural number of contrast peaks are found in an image picked up by the second imaging device 20 or where a peak is flat in shape, a focus difference between the front focus and the rear focus is adjusted, thus making it possible to detect a focus position of the sample S at high accuracy.
Further, where, as the optical path difference producing member of the present embodiment, there are used optical path difference producing members 21 (21A) composed of a flat-plate like glass member, the optical path difference producing member 21 can be made simple in structure. In this case, an edge part E of the flat plate member forms a shadow 23 of the second optical image at the imaging area 20 a of the second imaging device 20. Therefore, the first imaging region 22A and the second imaging region 22B are set so as to avoid the shadow 23, by which it is possible to secure a focus position of the objective lens 15 at high accuracy.
Further, in the above-described embodiment, the first imaging region 22A and the second imaging region 22B are set so as to be positioned on the front side of the sample S in the scanning direction on the imaging area 20 a with respect to the region 22C into which an optical image is made incident which is conjugate to the first optical image made incident into the first imaging device 18. Thereby, it is possible to obtain in advance a deviation direction of the focus position in relation to an imaging position of the first imaging device 18. The present invention shall not be limited to the above-described mode but may include a mode in which analysis is made for a deviation direction of the focus position at an imaging position of the first imaging device 18.
In the above-described embodiment, there is exemplified an apparatus for producing a virtual micro image. The image capturing apparatus of the present invention is, however, applicable to various types of apparatuses, as long as the apparatuses are those in which an image is captured by scanning a sample at a predetermined speed by a stage etc.

REFERENCE SIGNS LIST

1 . . . stage, 12 . . . light source, 14 . . . light guiding optical system, 15 . . . objective lens, 16 . . . beam splitter (light dividing unit), 18 . . . first imaging device (first imaging unit), 20 . . . second imaging device (second imaging unit), 20 a . . . imaging area, 21 (21A to 21D) . . . optical path difference producing member, 22A . . . first imaging region, 22B . . . second imaging region, 34 . . . focus control portion (focus control unit), 35 . . . region control portion (region control unit), 36 . . . objective lens control portion (objective lens control unit), E . . . edge part, L1 . . . first optical path, L2 . . . second optical path, M . . . image capturing apparatus, M1 . . . macro image capturing device, M2 . . . micro image capturing device, P . . . axis, S . . . sample.

Claims

1. An apparatus for capturing an image of a sample, the apparatus comprising:

a stage configured to support a sample;

an objective lens configured to face to the sample;

a stage control unit configured to move the stage at a moving speed;

a beam splitter optically coupled and configured to divide an optical image of at least a portion of the sample into a first optical image and a second optical image;

a first image sensor configured to capture the at least a portion of the first optical image;

a second image sensor configured to capture the at least a portion of the second optical image and provide image data;

a focus controller configured to analyze the image data so as to control a focus position of the objective lens based on the analysis result;

a region control unit of a computer system, configured to set, at an imaging area of the second image sensor, a first imaging region and a second imaging region so as to capture the at least the portion of the second optical image; and

wherein the region control unit is configured to reverse a position of the first imaging region and a position of the second imaging region on the imaging area.