JP2018116309A - Image acquisition device, image acquisition method, and microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve convenience and efficiency for a user, and acquire a z stack without missing of photographs of an observation object while reducing the number of z stack images.SOLUTION: The image acquisition device has an imaging element that acquires scattered light from a biological sample irradiated with illumination light. The imaging element is subjected to exposure at a first in-focus position and then subjected to exposure at a second in-focus position set with predetermined spacing with the first in-focus position, resulting in a pixel signal multiple-exposed at the first and second in-focus positions.SELECTED DRAWING: Figure 5

Description

  The present technology relates to an image acquisition device that acquires an image using a microscope, an image acquisition method, and a computer program.

  In pathological diagnosis, a high-definition biological sample image obtained by enlarging a biological sample such as a tissue section at a predetermined magnification is used. Therefore, a region where a biological sample is present is divided into a plurality of small regions, and the small region is enlarged and imaged at a predetermined magnification to obtain a divided image, and the plurality of divided images are combined to obtain a high-definition biological body. A microscope apparatus for generating a sample image has been proposed.

  Further, the same region of a biological sample is imaged at a plurality of focal positions at intervals of several μm to visualize each of them. A set of a plurality of microscope images picked up under different conditions of the focal positions is called a “Z stack” (for example, see Patent Document 1).

Special table 2008-500643

  For example, if a biological sample having a thickness of 10 μm is imaged at the focal positions with an interval of 1 μm, 10 imaging operations are required. That is, ten images are obtained for one small area. Further, when an optical system with a depth of focus of 1 μm is adopted, there are many opinions that an interval of 0.5 μm or less is appropriate because there is a possibility that an observation object may be missed at an interval of about 1 μm. However, as the focal position interval is reduced, the total capacity of the image data constituting one Z stack increases. Furthermore, when it becomes the data for one biological sample, it becomes a huge total capacity. This speeds up the replacement cycle of storage such as HDDs in the apparatus that stores image data captured by the microscope apparatus, and complicates maintenance and management, as well as data transmission and development processing from the microscope apparatus to the image data storage apparatus. This causes a decrease in imaging speed due to the bottleneck.

  An object of the present technology is to provide an image acquisition apparatus, an image acquisition method, and a microscope capable of solving such problems and improving convenience and efficiency for a user.

  In order to solve the above-described problem, an image acquisition device according to an embodiment of the present technology includes an image sensor that acquires scattered light obtained from a biological sample irradiated with illumination light, and the image sensor has a first focus. After the image sensor is exposed at a position, the image sensor is exposed at a second focus position set at a predetermined interval with respect to the first focus position, and the first focus position and the second focus position are exposed. A pixel signal subjected to multiple exposure at the in-focus position is obtained.

  The imaging element may obtain the pixel signal subjected to the multiple exposure by moving a stage on which the biological sample is placed.

The image sensor may obtain the multiple-exposed pixel signal by controlling the illumination light irradiation timing.
The illumination light may be strobe light emission.

  The image sensor may be a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal-Oxide Semiconductor) image sensor.

  According to another aspect of the present technology, an image acquisition method includes: exposing an image sensor at a first focus position with scattered light from a biological sample irradiated with illumination light; On the other hand, exposure is performed at a second in-focus position set at a predetermined interval to obtain a multiple-exposed pixel signal.

  An image acquisition device according to another aspect of the present technology includes a light source that irradiates a biological sample with illumination light, an imaging element that acquires scattered light obtained from the biological sample irradiated with the illumination light, and a first focus. After the image sensor is exposed at a position, the image sensor is exposed at a second focus position set at a predetermined interval with respect to the first focus position, and the first focus position and the first focus position are exposed. And a control unit that performs control so as to obtain a pixel signal subjected to multiple exposure at the in-focus position.

  As described above, according to the present technology, it is possible to improve convenience and efficiency for the user.

It is a figure which shows the typical Z stack imaging method. It is a figure which shows the imaging | photography method of this technique. It is a block diagram showing composition of an image acquisition device of a 1st embodiment concerning this art. It is a block diagram which shows the hardware constitutions of the data processing part in the image acquisition apparatus of FIG. It is a block diagram which shows the functional structure of the data processing part of FIG. It is a figure which shows the area | region of the imaging target by the image acquisition apparatus of FIG. FIG. 4 is a timing chart of the operation of each unit at the time of imaging a Z stack by the image acquisition device of FIG. 3. It is a figure which compares and shows the Z stack | stuck acquired with the image acquisition apparatus of this embodiment, and a typical Z stack | stuck. It is a figure which shows the relationship between the fixed focus image and average image of Z stack | stuck, and the imaging object area | region of FIG. It is a figure for comparing and showing an average image and a fixed focus image. It is a graph which shows the general defocus characteristic of a 540 nm green image. It is a graph which shows the defocus characteristic of the average image of the range of 4 micrometers. It is a timing chart which shows the modification 1 of the operation | movement at the time of imaging of Z stack. It is a timing chart which shows the modification 2 of the operation | movement at the time of imaging of Z stack. 12 is a timing chart showing a third modification example of the operation during imaging of the Z stack. 12 is a timing chart showing a fourth modification example of the operation during imaging of the Z stack.

Hereinafter, embodiments according to the present technology will be described with reference to the drawings.
<First Embodiment>
[1. Overview of the image acquisition apparatus of the present embodiment]
FIG. 1 is a diagram illustrating a typical Z stack imaging method.
For example, in the inspection of a thick biological sample such as a cell smear slide, a plurality of focal positions (Z1, Z2) can be found without missing an observation target such as bacteria 2 that may exist in the cell 1. ,..., Zn). A plurality of images captured at such a plurality of focal positions are called “Z stacks”.

  On the other hand, an objective lens having a numerical aperture as high as possible (for example, NA = 0.6 to 0.8) is used for the optical system of the microscope. The depth of focus of an optical system using an objective lens having such a numerical aperture is about 1 μm. In this case, by setting the interval between the Z stacks to 1 μm, it is theoretically possible to obtain a Z stack that covers the entire biological sample three-dimensionally without waste. However, for observation with less leakage, an image having a smaller interval, for example, 0.5 μm is desired.

  However, the smaller the Z stack interval, the greater the number of images in the Z stack and the overall data size. When the data size of the Z stack unit increases, it takes a long time to acquire an image, and increases the cost required for maintenance such as storage for storing the image and its replacement.

For example, as illustrated in FIG. 2, the image acquisition apparatus according to the present embodiment has an image sensor so that an average image covering the range can be obtained for each range divided by a position in a direction in which the focus can be moved. Is subjected to multiple exposure at a plurality of positions. Here, the length of the range is set to a length obtained by multiplying the depth of focus of the optical system using the objective lens by the number of exposure multiples, or less than the length.
More specifically, the image acquisition apparatus according to the present embodiment has, for example, a plurality of focal positions (Z1, Z2,... Zn) determined in advance based on the focal depth of an optical system using an objective lens. The average image is acquired by performing multiple exposure of the image sensor at a plurality of continuous focal positions, or by continuously exposing the image sensor across a plurality of continuous focus positions. As a result, it is possible to obtain a Z stack in which the number of images in the Z stack is reduced and there is no imaging omission of the observation target.
Details of the image acquisition apparatus of this embodiment will be described below.
Note that the present embodiment is an image acquisition device that acquires the above average image by performing multiple exposure of an image sensor at two consecutive focal positions.

[2. Configuration of image acquisition apparatus]
FIG. 3 is a diagram illustrating a configuration of the image acquisition apparatus 100 according to the present embodiment.

The image acquisition apparatus 100 includes a microscope 10 and a data processing unit 20.
The microscope 10 includes a stage 11, an optical system 12, a light source unit 13, an image sensor 14, a light source driving unit 15, a stage driving unit 16, and a camera control unit 17.

  The stage 11 has a surface on which a biological sample SPL such as a tissue section, a cell, or a chromosome to be imaged can be placed. The stage 11 is configured to be movable in a parallel direction (xy axis direction) and an orthogonal direction (z axis direction) with respect to the surface.

  In this embodiment, the biological sample SPL is fixed to the slide glass SG by a predetermined fixing method, and is stained as necessary. This staining includes not only general staining represented by HE (Hematoxylin-Eosin) staining, Giemsa staining, Papanicolaou staining, etc., but also fluorescence staining such as FISH (Fluorescence In-Situ Hybridization) and enzyme antibody method.

  An optical system 12 is disposed on one surface side of the stage 11, and a light source unit 13 is disposed on the other surface side of the stage 11.

  The light source unit 13 emits light under the control of the light source driving unit 15, and irradiates the biological sample SPL disposed on one surface of the stage 11 through an opening formed in the stage 11. To do. The light source unit 13 includes, as the light source 13A, a white LED (Light Emitting Diode) that outputs white light. The light source unit 13 includes a condensing lens 13B that converts light emitted from the light source 13A into substantially parallel light and uses it as illumination light for the biological sample SPL.

  The optical system 12 enlarges a part of the image in the biological sample SPL obtained by the illumination light to a predetermined magnification by the objective lens 12A and the imaging lens 12B. The image magnified by the objective lens 12A and the imaging lens 12B is formed on the imaging surface of the imaging element 14. As the image sensor 14, an image sensor capable of simultaneous exposure in all light receiving units corresponding to all pixels, for example, a CCD (Charge Coupled Device) image sensor, a CMOS (Complementary Metal-Oxide Semiconductor) image sensor, or the like is used. .

  The light source drive unit 15 has at least a drive circuit that supplies a constant drive current to the light source 13A in the light source unit 13 based on the strobe emission command S1 from the data processing unit 20 to cause the light source 13A to emit light.

  The stage drive unit 16 supplies the stage drive currents in the xyz three-axis directions for driving the stage 11 based on the stage control signal S2 from the data processing unit 20 to move the stage 11 in the three-axis directions. .

  The camera control unit 17 controls the image sensor 14 based on the exposure control signal S3 from the data processing unit 20. The camera control unit 17 performs A / D (Analog to Digital) conversion on a signal (RAW data) corresponding to each pixel read from the image sensor 14 and supplies the signal to the data processing unit 20.

  The data processing unit 20 performs development processing of raw data supplied from the camera control unit 17 of the microscope 10, stitching processing of development data, and the like to generate a biological sample image, which is generated as JPEG (Joint Photographic Experts Group) or the like. Encode and store the data in a predetermined compression format. In addition, the data processing unit 20 performs arithmetic processing for controlling the light source driving unit 15, the stage driving unit 16, and the camera control unit 17 based on a predetermined program.

[3. Configuration of data processing unit]
Next, the configuration of the data processing unit 20 will be described.
FIG. 4 is a block diagram illustrating a hardware configuration of the data processing unit 20.
The data processing unit 20 receives a CPU (Central Processing Unit) 21 that performs arithmetic control, a ROM (Read Only Memory) 22, a RAM (Random Access Memory) 23 that is a work memory of the CPU 21, and a command according to a user operation. An operation input unit 24, an interface unit 25, a display unit 26, a storage unit 27, and a bus 28 for connecting them together are provided.

  The ROM 22 stores programs for executing various processes. The microscope 10 is connected to the interface unit 25.

  A liquid crystal display, an EL (Electro Luminescence) display, a plasma display, or the like is applied to the display unit 26. For the storage unit 27, a magnetic disk represented by an HDD (Hard Disk Drive), a semiconductor memory, an optical disk, or the like is applied.

  The CPU 21 develops a program corresponding to a command given from the operation input unit 24 among the plurality of programs stored in the ROM 22, and appropriately controls the display unit 26 and the storage unit 27 according to the developed program. . Further, the CPU 21 appropriately controls each unit of the microscope 10 via the interface unit 25 in accordance with a program developed in the RAM 23.

[4. Biological sample image acquisition process]
Next, a biological sample image acquisition process in the image acquisition apparatus 100 of the present embodiment will be described.

  When the CPU 21 receives an acquisition command for the image of the biological sample SPL from the operation input unit 24, the CPU 21 develops a program corresponding to the acquisition command in the RAM 23.

  As shown in FIG. 5, the CPU 21 follows a program corresponding to an image acquisition command for the biological sample SPL, as shown in FIG. 5, a stage control unit 31 (movement control unit), an exposure control unit 32 (multiple exposure processing unit), and a strobe control unit 33 ( Multiple exposure processing unit), image acquisition unit 34, development processing unit 35, image compression unit 36, and image recording unit 37.

  For example, as shown in FIG. 6, the stage control unit 31 adjusts a region PR (hereinafter also referred to as a sample region) PR to be imaged of the biological sample SPL according to the magnification of the objective lens 12A and the imaging lens 12B. Assign to multiple small areas AR.

  The stage control unit 31 moves the stage 11 so that the region imaged by the image sensor 14 among the plurality of small regions AR is, for example, the upper left small region AR.

  Thereafter, as shown in FIG. 6, the processing for acquiring a plurality of images at a plurality of focal positions (Z1, Z2,... Zn) as a Z stack for the upper left small area AR is as follows. To be executed. A plurality of focal positions (Z1, Z2,... Zn) are determined based on the focal depth of the optical system using the objective lens. In the present embodiment, the focal position interval is the same as the focal depth of the optical system 12 including the objective lens 12A. However, in the present technology, the focal position interval may be equal to or smaller than the focal depth of the optical system.

  FIG. 7 is a timing chart of the operation of each unit of the image acquisition apparatus 100 when the Z stack is captured. The on / off timings of light irradiation, exposure of the image sensor 14 and movement of the stage 11 in the z-axis direction are shown in order from the top.

Multiple exposure at the first two focal positions Z1 and Z2 First, the stage control unit 31 sets the position of the stage 11 in the z-axis direction so that the first focal position Z1 is focused.
The exposure control unit 32 supplies an exposure control signal S3 to the camera control unit 17 so that the image sensor 14 in the microscope 10 is in an exposure enabled state that is an image capture state. Upon receiving the exposure control signal S3, the camera control unit 17 sets the image sensor 14 in a state where exposure is possible prior to light irradiation from the light source unit 13 (T1).

  After the image sensor 14 is ready for exposure, the strobe control unit 33 in the data processing unit 20 supplies a strobe emission command S1 to the light source driving unit 15 so that a constant amount of light is emitted from the light source unit 13. . Thus, simultaneous exposure (initial exposure) of all pixels of the image sensor 14 at the focal position Z1 is performed (T2).

  Here, typically, the value for each pixel of the image sensor 14 is captured by the camera control unit 17 and transmitted to the data processing unit 20, but in this embodiment, the exposure control unit 32 can expose the image sensor 14. Let the state continue.

  After the first exposure of the image sensor 14, the stage control unit 31 of the data processing unit 20 supplies the stage control signal S2 to the stage drive unit 16 so as to move the focal position to Z2 (T3). When the movement to the focal position Z2 is completed, the stage control unit 31 stops the stage 11 (T4).

  Thereafter, the strobe control unit 33 supplies a strobe emission command S1 to the light source driving unit 15 so that light is emitted from the light source unit 13 again. Thereby, the image sensor 14 is exposed (second exposure) at the focal position Z2 (T5).

  Here, in the light receiving portions of all the pixels of the image sensor 14, charges have already been accumulated by exposure at the focal position Z1. Therefore, by the exposure at the current focal position Z2, the charges obtained by photoelectric conversion at the two focal positions Z1 and Z2 are accumulated in the light receiving portions of all the pixels of the image sensor 14 in a state of being added for each pixel. Is done. That is, the image sensor 14 is double-exposed at the two focal positions Z1 and Z2, so that the images at the two focal positions Z1 and Z2 are combined as one average image.

  After the second exposure of the image sensor 14 (exposure at the focal position Z2) is completed, the exposure control unit 32 supplies a control signal S3 to the camera control unit 17 so as to capture data of all pixels from the image sensor 14. . In accordance with the control signal S3, the camera control unit 17 takes in voltage signals corresponding to the charges accumulated in the light receiving units of all the pixels of the image sensor 14 during a period from T6 to T7, and performs necessary signals such as A / D conversion. Data of all pixels generated by performing the processing is transmitted to the exposure control unit 32 of the data processing unit 20 as image RAW data. This completes the imaging of the average image by multiple exposure at the two focal positions Z1 and Z2.

Multiple exposure at the next two focal positions Z3 and Z4 Subsequently, imaging by multiple exposure is performed similarly for the next two focal positions Z3 and Z4. That is, when the capture of the image from the imaging device 14 that has been subjected to multiple exposure at the previous two focal positions Z1 and Z2 is completed, the exposure control unit 32 supplies the camera control unit 17 with the exposure control signal S3 again. The image sensor 14 is again exposed (T8).

  When the second exposure (exposure at the focal position Z2) of the image sensor 14 is completed (T6), the stage control unit 31 sends the stage control signal S2 to the next focal position Z3 so as to move to the next focal position Z3. 16 is supplied. When the movement to the next focal position Z3 is completed, the stage controller 31 stops the stage 11 (T9).

  When the image sensor 14 is ready for exposure and the movement to the next focal position Z3 is completed, the strobe control unit 33 causes the light source driving unit 15 to emit light from the light source unit 13 again. Supply. Thereby, the first exposure of the image sensor 14 at the next focal position Z3 is performed (T10).

  The exposure control unit 32 continues the exposure enabled state of the image sensor 14 even after the light irradiation from the light source unit 13 to the biological sample SPL is completed. When the emission of light from the light source unit 13 is completed, the stage control unit 31 of the data processing unit 20 supplies the stage control signal S2 to the stage driving unit 16 so as to move to the next focal position Z4. When the movement to the next focal position Z4 is completed, the stage control unit 31 stops the stage 11 (T11).

After that, the strobe control unit 33 supplies a strobe emission command S1 to the light source driving unit 15 so that light is emitted from the light source unit 13 again. As a result, the second exposure of the image sensor 14 at the next focal position Z4 is performed (T12). After the second exposure of the image sensor 14 (exposure at the focal position Z4) is completed, the exposure control unit 32 supplies a control signal S3 to the camera control unit 17 so as to capture data of all pixels from the image sensor 14. . In accordance with the control signal S3, the camera control unit 17 takes in voltage signals corresponding to the charges accumulated in the light receiving units of all the pixels of the image sensor 14 during a period from T13 to T14, and performs necessary signals such as A / D conversion. The digital data of all the pixels generated by performing the processing is transmitted to the exposure control unit 32 of the data processing unit 20 as image RAW data. This completes the acquisition of the average image by double exposure at the next two focal positions Z3 and Z4.
In the subsequent focal positions, the average image acquisition by double exposure is repeated in the same manner.

  The Z stack composed of a plurality of average images captured as described above has a smaller number of images than the Z stack obtained by a typical method in which the image sensor is exposed only once at each focal position. Significantly less. In addition, since each image is an average image by double exposure of the image sensor 14 at two focal positions, observation is performed if the focal depth of the optical system using the objective lens is equal to or greater than the interval between the two focal positions. It is possible to obtain a Z stack with no target imaging omission.

FIG. 8 is a diagram showing a comparison between an average image acquired by the image acquisition apparatus 100 of the present embodiment and a fixed focus image in a typical Z stack.
Each fixed focus image in a typical Z stack was obtained by imaging at respective focal positions Z1, Z2,..., Z10,... (See FIG. 1) with an imaging interval of 0.5 μm. It is an image. These fixed focus images are a part of BR in the image (small area AR in FIG. 6) imaged by the image sensor 14 as shown in FIG.

  On the other hand, the average image Z1-Z2 is an image obtained by double exposure at two focal positions Z1 and Z2. The average image Z3-Z4 is an image obtained by double exposure at two focal positions Z3, Z4. Average images Z5-Z6 are images obtained by double exposure at two focal positions Z5, Z6. The average images Z7 to Z8 are images obtained by double exposure at two focal positions Z7 and Z8. The average images Z9-Z10 are images obtained by double exposure at two focal positions Z9, Z10.

  As can be seen by comparing these average images and fixed focus images, the average image quality is slightly inferior to the corresponding fixed focus images due to double exposure, but there is no problem in observing biological samples. It can be said.

The average image may be not only an image obtained by double exposure at two consecutive focal positions Z3 and Z4 but also an image obtained by multiple exposure at three or more focal positions. In FIG. 8, an average image Z1-Z4 is an image obtained by multiple exposure at four focal positions Z1, Z2, Z3, and Z4. The average image Z5-Z8 is an image obtained by multiple exposure at the four focal positions Z5, Z6, Z7, and Z8. The average images Z9 to Z12 are images obtained by multiple exposure at the four focal positions Z9, Z10, Z11, and Z12.
Thus, it was confirmed that the average image obtained by the multiple exposure at the four consecutive focal positions can obtain an image quality with no problem in observing the biological sample. For reference, FIG. 10 shows a comparison between an average image obtained by multiple exposure at four consecutive focal positions and a fixed focus image at the four focal positions.

Next, the reason why the image quality of the average image obtained by the image acquisition apparatus 100 according to the present embodiment is sufficient for the biological sample observation application will be described.
FIG. 11 is a graph showing the defocus characteristics of a bright spot image obtained when 540 nm (green) light emitted from a fine light emitting point is observed with an objective lens of NA 0.8.
In this graph, the vertical axis is the count value (intensity count) indicating the brightness of each pixel for which information has been calculated, the horizontal axis is the pixels on the imager, and the lines in the graph are the optical axes for each defocus amount. The correlation between the deviation amount and the count value is shown. The count value (intensity count) is an index value of luminance (contrast). Thus, in the defocus characteristic of a 540 nm green image, it can be seen that the brightness (contrast) is drastically decreased when the defocus amount exceeds 2 μm.

  FIG. 12 is a graph showing the defocus characteristics of an average image in the range of 4 μm. A line indicated by a dotted line is a defocus characteristic of the fixed focus image, and a plurality of other solid lines are defocus characteristics of an average image in a range of 4 μm. That is, the average image A is an average image in the range from the in-focus position to 4 μm below the in-focus position, and the average image B is the average image and average image in the range from the in-focus position to 4 μm above the in-focus position. C is an average image in a range from 3 μm below the in-focus position to 1 μm above the in-focus position, and average image D is in a range from 1 μm below the in-focus position to 3 μm above the in-focus position. The average image E and the average image E are average images E ranging from a position 2 μm above the in-focus position to 2 μm below the in-focus position. Since the defocus characteristics of the average image A and the average image B are almost the same, they are shown by one solid line. Similarly, since the defocus characteristics of the average image C and the average image D are almost the same, these are also shown by one solid line.

  When FIG. 11 and FIG. 12 are compared, the luminance (contrast) of the average image in the range of 4 μm is almost the same as that when the defocus amount is 1 μm, which can be said to be sufficient for the observation of biological samples.

  As described above, according to the image acquisition apparatus 100 of the present embodiment, it is possible to obtain a Z stack in which there is no omission in imaging of an observation target with a smaller number of images while ensuring a practically sufficient image quality. As a result, it is possible to extend the replacement cycle of a storage such as an HDD in the apparatus that accumulates the captured image data, thereby reducing the complexity of maintenance management. In addition, the image acquisition speed can be improved.

<Modification 1>
Next, a modification of the above embodiment will be described.
FIG. 13 is a timing chart showing Modification Example 1 of the operation at the time of imaging the Z stack.
In the image acquisition device of the first modification, in the Z stack imaging operation by the image acquisition device 100 of the above-described embodiment, the stage 11 is continuously moved, that is, the focal position is continuously moved. . It is desirable that the moving speed of the stage 11 is constant at least during exposure.

  According to this method, the time required for stabilizing the stage 11 can be eliminated, so that the overall time for Z stack imaging can be reduced. Note that the operation of the image acquisition device of the first modification is basically the same as that of the image acquisition device 100 of the above embodiment except that the stage 11 is not stopped. Also by this modification 1, the effect of said embodiment can be acquired similarly.

<Modification 2>
FIG. 14 is a timing chart showing a second modification example of the operation during imaging of the Z stack.
In the image acquisition apparatus according to the second modification, the light irradiation timing is set so that the stage 11 is continuously moved and the image sensor 14 is continuously exposed across a plurality of continuous focal positions. Other operations of the image acquisition apparatus of the second modification are basically the same as those of the image acquisition apparatus of the first modification. The effect of the above-described embodiment can be obtained in the same manner in the second modification. In addition, by continuously exposing the imaging element 14 across a plurality of consecutive focal positions, the risk of overlooking due to imaging omission of the observation target can be further reduced as compared with the first modification. it can.

<Modification 3>
FIG. 15 is a timing chart showing a third modification example of the operation during imaging of the Z stack.
In the image acquisition device of the third modification, while the image sensor 14 is continuously exposed across a plurality of continuous focal positions, the light irradiation of the light source unit 13 is turned on and off at a constant cycle shorter than the focal position interval. It is made to switch repeatedly. Also in the third modification, the effects of the image processing apparatus 100 of the above-described embodiment and the image acquisition apparatus of the second modification can be obtained in the same manner. According to the third modification, the exposure time can be adjusted by selecting the on-duty ratio of light irradiation.

<Modification 4>
FIG. 16 is a timing chart showing Modification Example 4 of the operation at the time of imaging the Z stack.
The image acquisition apparatus according to the fourth modification continuously synchronizes the light irradiation timing from the light source unit 13 and the movement timing of the stage 11, and continuously switches the movement and stop of the stage 11 at every Z stack interval. The exposed position is continuous between the ranges (see FIG. 2).

  That is, the stage control unit 31 controls the stage 11 so as to move the focal position from Z1 to Z2 in one exposure time. The stage controller 31 stops the stage 11 when one exposure is completed, and controls the stage 11 to move the focal position from Z2 to Z3 during the next exposure time. According to the fourth modification, since it is possible to perform imaging that covers the entire range in the z-axis direction, compared with the above-described embodiment and the modification, the risk of oversight due to the imaging omission of the observation target is increased. It can reduce more reliably.

  As mentioned above, although the image acquisition apparatus which images a transmitted illumination image has been described, it is needless to say that the present technology can be similarly applied to an apparatus that captures a fluorescent image using a dark field light source.

SPL ... biological sample 10 ... microscope 20 ... data processing unit 11 ... stage 12 ... optical system 12A ... objective lens 12B ... imaging lens 13 ... light source unit 13A ... light source 13B ... condensing lens 14 ... imaging element 15 ... light source drive unit 16 ... stage drive unit 17 ... camera control unit 21 ... CPU
22 ... ROM
23 ... RAM
DESCRIPTION OF SYMBOLS 24 ... Operation input part 31 ... Stage control part 32 ... Exposure control part 33 ... Strobe control part 34 ... Image acquisition part 100 ... Image acquisition apparatus

Claims (10)

Comprising an imaging device for obtaining scattered light obtained from a biological sample irradiated with illumination light;
The image sensor exposes the image sensor at a first focus position, and then exposes the image sensor at a second focus position set at a predetermined interval with respect to the first focus position. An image acquisition device for obtaining pixel signals subjected to multiple exposure at the first in-focus position and the second in-focus position. The image acquisition device according to claim 1,
The image acquisition device, wherein the imaging device obtains the multiple-exposed pixel signal by moving a stage on which the biological sample is placed. The image acquisition device according to claim 1,
The image acquisition device, wherein the imaging device obtains the multiple-exposed pixel signal by controlling the illumination light irradiation timing. The image acquisition device according to claim 3,
The image acquisition device, wherein the illumination light is strobe light emission. The image acquisition device according to any one of claims 1 to 4,
The image pickup device is an image acquisition device in which the image pickup device is a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal-Oxide Semiconductor) image sensor. The image acquisition device according to claim 1,
The image acquisition device, wherein the illumination light is transmitted illumination light or light from a dark field light source that obtains a fluorescent image. Comprising an imaging device for obtaining scattered light obtained from a biological sample irradiated with illumination light;
The image sensor exposes the image sensor at a first focus position, and then exposes the image sensor at a second focus position set at a predetermined interval with respect to the first focus position. A microscope for obtaining a pixel signal subjected to multiple exposure at the first in-focus position and the second in-focus position. After the imaging device is exposed at the first in-focus position by the scattered light from the biological sample irradiated with the illumination light, the second in- stance set with a predetermined interval with respect to the first in-focus position. An image acquisition method for obtaining a pixel signal subjected to multiple exposure by exposing at a focus position. Comprising an imaging device for acquiring scattered light obtained from a subject irradiated with illumination light;
The image sensor exposes the image sensor at a first focus position, and then exposes the image sensor at a second focus position set at a predetermined interval with respect to the first focus position. An image acquisition device for obtaining pixel signals subjected to multiple exposure at the first in-focus position and the second in-focus position. A light source for illuminating a biological sample with illumination light;
An imaging device for obtaining scattered light obtained from the biological sample irradiated with the illumination light;
After the imaging element is exposed at the first focus position, the image sensor is exposed at a second focus position set at a predetermined interval with respect to the first focus position. An image acquisition apparatus comprising: a control unit that performs control so as to obtain a pixel signal subjected to multiple exposure at a focal position and the second in-focus position.