JP2010091739A - Image pickup device including automatic focusing device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an image pickup device that achieves shortening of total photographing time by performing automatic focusing including the time excepting an exposure time, that is, the time to transfer an image from an image capturing means to an image recorder. <P>SOLUTION: The image pickup device includes the image capturing means, an image storage means, an objective lens 4, a sample 1, a stage 3 on which the sample is fixed, a means for switching an optical element, a means capable of relatively changing a distance between the sample and the objective lens, a light source 10, and the automatic focusing device 6, and automatically repeatedly picks up the image of the sample by using an automatic focusing method for performing focusing adjustment by changing a relative position of the objective lens to the sample. All or a portion of the time to transfer the image from the image capturing means to the image storage means is included in an automatic focusing operation period. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an optical apparatus used when observing a state of a biological tissue or the like in the fields of medicine, biology, and the like, for example, an image capturing apparatus including an automatic focusing device.

In such an image pickup apparatus, an image is automatically taken under a plurality of conditions in one field of view, or a large number of images are automatically taken in a plurality of areas, and images taken in this way are taken. Analysis from In this case, the dimensions of the main body of the device will change due to changes in imaging conditions such as objective lens replacement, movement of the imaging position, or changes in the shooting environment, so the focus position will change even if the focus position is set first. End up.
Therefore, in order to automatically and repeatedly perform a plurality of shootings, a method of focusing on an imaging position using an automatic focusing device is used.

  For example, in Patent Document 1, the imaging result of the illumination light image is acquired from the imaging unit, the focus evaluation is performed, and at least one of the objective lens and the specimen is driven according to the focus evaluation of the illumination light image, thereby obtaining a fluorescent image. Describes a fluorescence microscope provided with a focus control unit that focuses the image on the imaging surface.

Further, Patent Document 2 includes a drive stage, and focusing is performed based on the reflected light that has passed through the half-side region on the opposite side of the pupil of the objective lens while the sample is moved to a specific position by the drive stage. The sample is observed by the reflected light reflected by the sample out of the light incident on the sample through the objective lens from the light source detected by the two-dimensional photodetector after the sample has moved to a specific position. An optical microscope is described.
JP 2006-11149 A JP 2006-163122 A

  However, in the method described in Patent Document 1, the focus is not changed or the optical element is not changed under the following shooting conditions during recording of the shot image, so that the efficiency is lowered and the total shooting time is long. There is a problem of becoming.

  In the method described in Patent Document 2, the focus position is adjusted while the stage is moving. However, since the change time of the optical element is not included in the time of the automatic focus adjustment, the total photographing is still performed. There is a problem that the time becomes longer.

  By the way, when shooting is performed using a cooled CCD camera having 1.4 million pixels (1360 × 1024 pixels), the time required for transfer from the cooled CCD camera to the image storage device is, for example, 0.8 seconds. When the resolution of the image is further determined, the image transfer time when the image is taken with a 1100 pixel (4008 × 2672 pixel) cooled CCD camera is, for example, 3.5 seconds. In addition, it is conceivable that a camera with higher definition will be developed in the future, and the transfer time may be further increased. Although it is conceivable that the transfer speed is slightly increased by the improvement, the transfer time is always generated.

  When observing a state of a biological tissue or the like in a field such as medicine or biology, a cell culture container in which a plurality of wells are formed in a container called a microplate is used. For example, in the case of observing one well of a microplate in which 96 wells are formed using a 20 × objective lens, it is necessary to photograph 200 times in order to photograph the entire well. There is.

  For example, when taking a picture using a camera with 1.4 million pixels, it takes 1.5 seconds for autofocusing and 1.5 seconds for the exposure time. Therefore, when autofocusing is performed after image transfer, it takes 3 seconds from focusing to completion of shooting in one shooting, and 0.8 seconds are required to transfer to the image storage device. In addition, if two colors are dyed at one place and the filter is exchanged to perform twice photographing, the photographing time becomes 3 seconds × 2 times 6 seconds. As a result, including the image transfer time, (3 + 0.8 + 3 + 0.8) seconds × 200 locations × 96 wells takes about 40.5 hours. FIG. 5 shows a partial sequence diagram of this process.

  Further, when photographing using a camera with 11 million pixels, it takes 1.5 seconds for autofocusing and 1.5 seconds for exposure time. Therefore, when autofocus is performed after image transfer, it takes 3 seconds from focusing to completion of shooting in one shooting, and 3.5 seconds are required to transfer to the image storage device. . In addition, if two colors are dyed at one place and the filter is exchanged to perform twice photographing, the photographing time becomes 3 seconds × 2 times 6 seconds. As a result, including the image transfer time, it takes (3 + 3.5 + 3 + 3.5) seconds × 200 locations × 96 wells = about 69 hours. A partial sequence diagram of this step is shown in FIG.

  In this way, even when a single microplate is shot, the total number of shots is large, so autofocusing is performed each time and image transfer is performed each time. It will take a few days to complete.

  Therefore, it is desired to shorten the photographing time. Here, considering the shooting cycle, if an operation that can be performed at the same time is performed during the auto-focusing operation period, which normally takes about 3 seconds, it is necessary for the shooting. Time can be shortened. As a result of examining the operation that can be performed at the same time as the automatic focusing operation period, it was found that the image transfer from the image capturing unit to the image storage unit can be performed. It was also found that the optical element can be replaced. These operations are basically performed independently of the autofocus operation.

Here, for example, when autofocusing is performed during image transfer using a camera with 1.4 million pixels, 0.8 seconds required to transfer to the image storage device is not necessary. 6 seconds are required if two colors are dyed at one location and the filter is replaced and taken twice, so the total imaging time, including the image transfer time, is approximately 32 hours at 6 seconds × 200 locations × 96 wells. . FIG. 7 shows a partial sequence diagram of the steps in this case.
Therefore, by including the autofocusing time during image transfer, it can be shortened by 8.5 hours compared to the case of performing autofocusing after conventional image transfer.

In addition, using an 11 million pixel camera and autofocusing during image transfer takes 3.5 seconds + 1.5 seconds for 5 seconds from focusing to the end of shooting. Take it. Therefore, if two colors are dyed at one place and the filter is exchanged and the picture is taken twice, the total photography time including the image transfer time is 10 seconds × 200 places × 96 wells = about 53 hours. FIG. 8 shows a partial sequence diagram of the steps in this case.
Therefore, by including the autofocusing time during image transfer, it can be shortened by 17 hours compared to the case of performing autofocusing after conventional image transfer.

  In other words, if autofocusing and image transfer are performed at the same time, which is conventionally performed in individual time zones, the time required for the total photographing time can be shortened accordingly. Also, the time required for the entire photographing time can be shortened by replacing the optical element at the same time as the automatic focusing. It was noted that autofocusing, image transfer, and exchange of optical elements are operations capable of parallel processing.

  Therefore, the present invention has been made in view of the above problems, and by performing autofocusing including the time excluding the exposure time, that is, the time during which the image is transferred from the image capturing means to the image recording apparatus, An object of the present invention is to provide an image pickup apparatus capable of shortening the total shooting time. It is another object of the present invention to provide an image pickup apparatus that further shortens the total shooting time by including the replacement time of the optical element during autofocusing.

  In order to achieve the above object, an image pickup apparatus provided with the autofocus device of the present invention includes an image capturing means, an image storage means, an objective lens, a specimen, a stage for fixing the specimen, and an optical device. A means for switching elements; a means capable of relatively changing a distance between the specimen and the objective lens; a light source; and an autofocus device; and changing a relative position between the specimen and the objective lens. In the imaging apparatus that automatically and repeatedly images the specimen using an autofocus method that performs focusing adjustment, all or part of the image transfer time from the image capturing unit to the image storage unit is automatically It is characterized by being included in the focusing operation period.

  In the image pickup apparatus provided with the autofocus device of the present invention, it is preferable that the exposure time of the specimen is not included in the autofocus operation period.

  In the image pickup apparatus provided with the autofocus device of the present invention, it is preferable that the replacement time of the optical element is included in the autofocus operation period.

  In the image pickup apparatus provided with the autofocus device of the present invention, the specimen is preferably a well plate or a slide glass.

  In the image pickup apparatus provided with the autofocus device of the present invention, it is preferable that the stage is an electric stage that can freely move the sample on the XY plane.

  In the image pickup apparatus provided with the autofocus device of the present invention, it is preferable that the image capturing means is a cooled CCD camera.

  In the image pickup apparatus provided with the autofocus device of the present invention, the optical element is preferably an excitation filter, a dichroic mirror, and an absorption filter.

  Further, in the image pickup apparatus provided with the autofocus device of the present invention, the autofocus device irradiates the sample with laser light through the objective lens, and reflects the reflected light through the objective lens. It is preferable to use a pupil division method in which the light is received by the light receiving unit and the focus is focused based on the calculation result.

  Starting the focusing operation immediately after the end of exposure of the CCD camera, that is, the time during transfer to the image storage device, led to a reduction in the total photographing time. Moreover, the efficiency was improved by exchanging the excitation filter, the dichroic mirror, and the absorption filter constituting the optical element during the autofocusing time, which further reduced the time.

Next, an outline and a control procedure will be described prior to a detailed description of an image pickup apparatus provided with the autofocus device of the present invention.
An image pickup apparatus provided with the autofocus device of the present invention includes an image capturing means, an image storage means, an objective lens, a specimen, a stage for fixing the specimen, a means for switching optical elements, a specimen, Means capable of relatively changing the distance of the objective lens, a light source, and an autofocus device are provided.

In the configuration of the image pickup apparatus of the present invention, a CCD camera is used as the image capturing means, a computer is used as the image recording apparatus, and a revolver and stage of a fluorescence microscope are used as means for relatively changing the distance between the specimen and the objective lens. The revolver has an objective lens and the stage has a specimen. A filter changer is used as the optical element switching means.
As the specimen, a multi-well plate, a slide glass or a dish in which cells labeled with fluorescence are cultured is used.
A white illumination or LED light source is used as the light source, and the light illuminated from the light source passes through the optical element, and the sample is irradiated with excitation light through the objective lens to emit fluorescence. The fluorescence is exposed by a CCD camera.

  In addition, an image pickup apparatus equipped with the autofocus device of the present invention automatically and repeatedly samples a sample using an autofocus method that adjusts the focus by changing the relative position of the sample and the objective lens. I do.

The principle of the automatic focusing device will be described. Laser light emitted from a semiconductor laser is cut in half by a slit and converted into parallel light by a collimating lens. After that, only the P-polarized light component is transmitted through a PBS (Polarization Beam Splitter), polarized by 45 ° by a λ / 4 plate, reflected by the objective lens through the objective lens, and again polarized by 45 ° by the λ / 4 plate. It passes through the collimating lens and forms an image on the light receiving sensor.
The light receiving sensor is a two-divided photodiode (A region and B region) arranged around the optical axis.
The signal that enters the light receiving sensor performs the calculation of (A−B) / (A + B). The value is called an evaluation function value (Ef value) and is used for focusing determination. The position where the distance between the objective lens and the sample is relatively changed and the Ef value becomes 0 is determined as the in-focus position.

  The laser irradiated using this is irradiated to the specimen through the objective lens, and the reflected light returns to the light receiving unit of the autofocus device again through the objective lens, and the in-focus position is specified by the calculation result.

  In the image pickup apparatus provided with the autofocus device of the present invention, all or part of the image transfer time from the image capturing means to the image storage means is included in the autofocus operation period.

By transferring the image captured by the CCD camera during the auto-focusing operation period for the next shooting, a unique time for image transfer becomes unnecessary, and as a result, the total shooting time can be shortened. In addition, the start time of the image transfer and the start time of the auto-focusing operation are basically the same period, but are not necessarily the same period.
When the image transfer time is longer than the autofocus operation period, this long part may not be included in the autofocus operation period. Even in that case, the length of the auto-focusing operation period in the image transfer time is shortened.

  In the image pickup apparatus provided with the autofocus device of the present invention, preferably, the specimen exposure time is not included in the autofocus operation period.

  During the automatic focusing operation period, the specimen is not exposed by the CCD camera. On the other hand, image transfer after the end of exposure is performed during the autofocus operation period for the next shooting.

  In the image pickup apparatus provided with the autofocus device of the present invention, preferably, the optical element replacement time is included in the autofocus operation period.

  The optical element can be replaced even during the autofocus operation period. Therefore, the total imaging time can be shortened by exchanging the optical elements simultaneously during the automatic focusing operation period.

  In the image pickup apparatus provided with the autofocus device of the present invention, the specimen is preferably a well plate or a slide glass.

  Since the image pickup apparatus of the present invention is mainly used for observing the state of a biological tissue or the like in the field of medicine, biology, etc., such a sample is used. It is particularly suitable for photographing such specimens.

  In the image pickup apparatus including the autofocus device of the present invention, the stage is preferably an electric stage that can freely move the specimen on the XY plane.

  As described above, the image pickup apparatus of the present invention is mainly used for observing the state of a biological tissue or the like in the fields of medicine, biology, etc., and a well plate, a slide glass or the like is used as a specimen. Therefore, an electric stage is used to automatically and repeatedly take a large number of times.

  In the image pickup apparatus provided with the autofocus device of the present invention, preferably, the image capturing means is a cooled CCD camera.

  Since many times of shooting are performed over a long period of time, a cooled CCD camera is used to perform shooting in a stable state.

  In the image pickup apparatus provided with the autofocus device of the present invention, the optical elements are preferably an excitation filter, a dichroic mirror, and an absorption filter.

  Such an optical element is necessary for fluorescence observation. In order to facilitate exchange according to the wavelength to be excited, they are usually integrated.

  In the image pickup apparatus provided with the autofocus device of the present invention, the autofocus device preferably irradiates the specimen with laser light through the objective lens and receives the reflected light through the objective lens. This is a pupil division method in which the light is received by a unit and focused by the calculation result.

  This method is suitable for autofocusing of the image pickup apparatus of the present invention in view of the time and accuracy required for focusing.

  Next, the control procedure of the image pickup apparatus provided with the autofocus device of the present invention will be described based on the sequence diagram of FIG.

First, when the laser of the autofocus device is turned on, the focus position is detected by calculation, the revolver is driven, the distance between the objective lens and the sample is controlled, and the objective lens is moved to the focus focus position.
When the objective lens is moved to the focus position, the laser of the autofocus device is turned off.

  Next, the shutter of the light source is opened, the sample is irradiated with excitation light, and the CCD camera is exposed.

When the exposure of the CCD camera is completed, the shutter of the light source is closed, and the image stored in the CCD camera is transferred to a computer which is an image storage device.
Further, in order to perform the next imaging immediately after the exposure is stopped, the laser of the autofocus device is turned on, and the focus position is detected.
While the in-focus position is detected, the filter changer is driven and the filter is replaced.
Upon completion of the operation of the filter changer, the shutter of the light source is opened and exposure is performed by the CCD camera.

  As a result, since the automatic focus adjustment is performed immediately after the exposure of the CCD camera, that is, including the image transfer time, the process is not wasted and the total time is reduced. Further, the efficiency is further improved by including the replacement of the optical element in the time of the automatic focus adjustment.

  Next, an embodiment of an image pickup apparatus provided with the autofocus device of the present invention will be described with reference to FIGS. FIG. 2 is an explanatory diagram of an image pickup apparatus provided with the autofocus device of the present invention. FIG. 3 is a sequence diagram showing details of a control method of the image pickup apparatus shown in FIG.

First, an image pickup apparatus provided with the autofocus device of the present invention will be described based on FIG.
In the image pickup apparatus provided with the autofocus device of the present invention, the cooled CCD camera 11 is used as the image capturing means. A computer is used as the image recording device 15. As the specimen 1, a well plate or a slide glass is used. As a means for relatively changing the distance between the specimen 1 and the objective lens 4, a revolver 16 of a fluorescence microscope and an electric XY stage 3 are used. An objective lens 4 is mounted on the revolver 16, and a specimen 1 is mounted on the electric XY stage 3. A filter changer 17 is used as the optical element switching means.

  As the light source 10, white illumination or an LED light source is used. The light illuminated from the light source 10 passes through the optical element, and the sample 1 is irradiated with the excitation light 13 through the objective lens 4 to emit fluorescence. Then, the fluorescence is exposed by the cooled CCD camera 11. The optical element includes an excitation filter 8, a second dichroic mirror 7, and an absorption filter 9, which are integrated for convenience of replacement by the filter changer 17.

  The laser beam 12 emitted from the autofocus device 6 is folded back by the first dichroic mirror 5 and reflected by the slide glass 1 through the objective lens 4. The reflected light is turned back by the first dichroic mirror 5 through the objective lens 4, returned to the automatic focusing device 6 and received by the light receiving sensor, and the in-focus position is determined by the calculation result.

  Then, the control device 14 performs integrated control of the electric XY stage 3, the autofocus device 6, the light source 10, the light source shutter 10 ', the cooling CCD camera 11, the image storage device 15, and the revolver 16. ing. Based on the calculation result of the automatic focusing device 6, the relative position between the sample 1 and the objective lens 4 is changed, and focusing adjustment is performed. The control device 14 performs autofocusing, shooting, image transfer, and optical element replacement in a predetermined order.

Next, an example of a control method of the image pickup apparatus provided with the above-described autofocus device of the present invention will be described with reference to FIG.
In this embodiment, the optical element is changed for the same imaging area, and images are taken twice under the first imaging condition and the second imaging condition.

As a specimen, cells were cultured in the imaging range 2 on the slide glass 1, and the nucleus was stained with DAPI and the cytoplasm was stained with GFP.
The slide glass 1 was attached to the electric XY stage 3, the imaging conditions were set, and a program was set in the control device 14 so as to image 3 × 3 imaging areas (2 a 1 to 2 c 3) with respect to the imaging range 2. The control device 14 controls the electric XY stage 3, the autofocus device 6, the light source 10, the light source shutter 10 ', the cooling CCD camera 11, the image storage device 15, and the revolver 16.

  First, automatic focus adjustment for photographing a nucleus is performed as a first imaging condition. The laser beam 12 emitted from the automatic focusing device 6 is reflected by the first dichroic mirror 5 and reflected by the slide glass 1 through the objective lens 4. The reflected light is turned back by the first dichroic mirror 5 through the objective lens 4, returned to the automatic focusing device 6 and received by the light receiving sensor, and the in-focus position is determined by the calculation result. Then, the revolver 16 is moved in the Z direction, the relative distance between the objective lens 4 and the slide glass 1 is changed, and is adjusted to the in-focus position.

  During the automatic focus adjustment, the filter changer 17 replaces the excitation filter 8, the second dichroic mirror 7, and the absorption filter 9. The excitation filter 8, the second dichroic mirror 7, and the absorption filter 9 that are optical elements are integrated to facilitate replacement according to the wavelength to be excited.

When the focusing is completed, the laser beam 12 of the automatic focusing device 6 is stopped.
At the same time, the light source shutter 10 ′ of the light source 10 is opened, and the irradiated light passes only a specific wavelength by the excitation filter 8, is turned back by the second dichroic mirror 7, and passes through the objective lens 4 to the first imaging area. (2a1) is irradiated.

  The reflected light passes through the absorption filter 9 via the objective lens 4 and is imaged by the cooled CCD camera 11 with a specified exposure time.

  Next, in order to capture cytoplasm under the second imaging condition, immediately after the exposure is completed, that is, at the same time as the transfer of the image under the first imaging condition from the cooled CCD camera 11 to the image storage device 15 is started, The laser beam 12 is emitted from the focusing device 6 and focusing for shooting under the second imaging condition is performed. During focusing, the filter changer 17 exchanges the excitation filter 8, the second dichroic mirror 7 and the absorption filter 9 constituting the optical element.

When the focusing is completed, the laser beam 12 of the automatic focusing device 6 is stopped.
At the same time, the light irradiated with the light source shutter 10 ′ being opened passes only a specific wavelength by the excitation filter 8, is turned back by the second dichroic mirror 7, and the first imaging area (2 a 1) is passed through the objective lens 4. Irradiated.
The reflected light passes through the absorption filter 9 via the objective lens 4 and is imaged by the cooled CCD camera 11 with a specified exposure time.

  When the exposure under the first imaging condition and the second imaging condition in the first imaging area (2a1) is completed, the slide glass 1 is moved to the next imaging area (2a2) by the electric XY stage 3. Then, the above operation is automatically repeated until the final imaging area (2c3) is imaged.

Next, an example is shown in which the 96-hole microplate 1 shown in FIG. 4 is prepared as a sample and taken by the image pickup apparatus shown in FIG.
In each well 2, the cell nucleus stained with DAPI and the cytoplasm stained with GFP is fixed to the bottom of the well 2. The microplate 1 has a flat bottom surface, is transparent, and can be observed from the bottom surface.

  The microplate 1 was attached to the electric XY stage 3 of the image photographing apparatus. An exposure time of 1.5 seconds and a 20 × objective lens are set, and 14 × 14 shooting areas (2a1) are arranged at a pitch of 400 μm in the horizontal direction and a pitch of 300 μm in the vertical direction so that the whole image is taken for each well 2. ~ 2n14) was set.

The control device 14 controls the electric XY stage 3, the autofocus device 6, the light source 10, the light source shutter 10 ′, the cooling CCD camera 11, the image recording device 15, and the revolver 12.
For the cooled CCD camera 11, BS41 made by Bitlan Co. was used. The specification is 1.4 million pixels (1326 × 1024 pixels), and it takes 0.8 seconds to transfer one image to the image recording device 15.

  First, the electric XY stage 3 is moved, and the well 2 of the microplate 1 is moved to the first imaging location. At the same time, the revolver 16 is rotated and controlled so that the 20 × objective lens 4 enters the optical path.

First, in order to photograph nuclei stained with DAPI, the filter changer 17 exchanges the excitation filter 8, the second dichroic mirror 7, and the absorption filter 9 which are optical elements. The excitation filter 8, the second dichroic mirror 7, and the absorption filter 9 are integrated in order to facilitate exchange according to the wavelength to be excited.
Note that Olympus filter cube U-MWG2 was used as an optical element for observing nuclei stained with DAPI.

  At the same time, the laser beam 12 of the automatic focusing device 6 is turned on and focusing is performed. The laser beam 12 emitted from the autofocus device 6 is folded back by the first dichroic mirror 5, reflected by the upper surface of the microplate 1, and folded back by the first dichroic mirror 5 again, and the light receiving sensor of the autofocus device 6. Return to.

  The light receiving sensor is a two-divided photodiode (A region and B region) arranged around the optical axis. The signal that enters the light receiving sensor performs the calculation of (A−B) / (A + B). The value is called an evaluation function value (Ef value) and is used for focusing determination. The position where the distance between the objective lens and the sample is relatively changed and the Ef value becomes 0 is determined as the in-focus position. The position where the Ef value is 0 is different from the in-focus position because it is the upper surface position of the microplate 1. Therefore, an offset value is set by a predetermined value from the upper surface of the microplate 1 to the position to be observed, and the distance is automatically set to the observation position.

  Then, the distance between the objective lens 4 and the microplate 1 is relatively changed by the revolver 16 and is adjusted to the in-focus position.

  When the focusing is completed, the laser beam 12 of the automatic focusing device 6 is stopped.

  Next, the light source shutter 10 ′ of the light source 10 is opened, and the irradiated light passes only a specific wavelength by the excitation filter 8, is turned back by the second dichroic mirror 7, and is passed through the objective lens 4 to the first imaging area ( 2a1) is irradiated.

  The reflected light passes through the absorption filter 7 via the objective lens 4 and is exposed by the cooled CCD camera 11.

The image accumulated by the cooled CCD camera 11 is transferred to the image storage device 15.
At the same time, immediately after the exposure is completed, that is, at the same time as the transfer of the image from the cooled CCD camera 11 to the image storage device 15 is started in order to capture the cytoplasm stained with GFP, the laser beam from the autofocus device 6 12 is irradiated and focusing is performed. At the same time, the filter changer 17 exchanges the excitation filter 8, the dichroic mirror 7, and the absorption filter 9.
Note that Olympus filter cube U-MGFPHQ was used as an optical element for imaging the cytoplasm stained with GFP.

  When the focusing is completed, the laser beam 12 of the automatic focusing device is stopped. Then, the light source shutter 10 ′ is opened and exposure is performed by the cooled CCD camera 11.

  When the exposure of the imaging area (2a1) is completed, the image accumulated by the cooled CCD camera 11 is transferred to the image storage device 15. At the same time, the microplate 1 is moved by the electric XY stage 3 to photograph the next imaging area (2a2). At the same time, the laser beam 12 is emitted from the automatic focusing device 6 and focusing is performed. At the same time, the filter changer 17 exchanges the excitation filter 8, the second dichroic mirror 7, and the absorption filter 9 as optical elements in order to photograph the nucleus stained with DAPI in the next imaging area (2a2).

  When the focusing is completed, the laser beam 12 of the automatic focusing device is stopped. Then, the light source shutter 10 ′ is opened and exposure is performed by the cooled CCD camera 11.

The above operation is repeated, all the images in the well 2 up to the last imaging area (2n14) are imaged, and all 96 wells 2 of the microplate 1 are imaged.
As a result, the time required for photographing all 96 wells 2 of the microplate 1 was 32 hours. Further, when the autofocus operation was performed after the image transfer, it took 40.5 hours, so in this embodiment the time was reduced to 8.5 hours.

It is a sequence diagram which shows the control procedure of an image pick-up device provided with the autofocus device of the present invention. It is explanatory drawing of the image pick-up device provided with the autofocus device of the present invention. It is a sequence diagram which shows the detail of the control method of the image imaging device shown in FIG. It is explanatory drawing which shows an example of a microplate. It is a sequence diagram which shows a part of process in the case of performing an autofocus after an image transfer using a 1.4 million pixel camera. It is a sequence diagram which shows a part of process in the case of performing an autofocus after an image transfer using a 11 million pixel camera. It is a sequence diagram which shows a part of process in the case of using a 1.4 million pixel camera and performing autofocusing during image transfer. It is a sequence diagram which shows a part of process in the case of performing autofocusing during image transfer using a 11 million pixel camera.

Explanation of symbols

1 Slide glass (microplate, specimen)
2 Imaging range (well)
2a1 to 2n14 Image pickup area 3 Electric XY stage 4 Objective lens 5 First dichroic mirror 6 Auto focus device 7 Second dichroic mirror 8 Excitation filter 9 Absorption filter 10 Light source 10 ′ Light source shutter 11 Cooling CCD camera 12 Laser light 13 Excitation light 14 Control device 15 Image storage device 16 Revolver 17 Filter changer

Claims (8)

  Image capturing means, image storage means, objective lens, specimen, stage for fixing the specimen, means for switching optical elements, and means capable of relatively changing the distance between the specimen and the objective lens And a light source and an autofocus device, and automatically and repeatedly image the sample using an autofocus method that adjusts the focus by changing the relative position of the sample and the objective lens. An image provided with an autofocus device characterized in that in the imaging device to be performed, all or part of the image transfer time from the image capturing means to the image storage means is included in the autofocus operation period. Imaging device.   2. The image capturing apparatus with an autofocus device according to claim 1, wherein an exposure time of the specimen is not included in the autofocus operation period.   3. The image pickup apparatus provided with the autofocus device according to claim 1, wherein the replacement time of the optical element is included in the autofocus operation period.   The image pickup apparatus provided with the automatic focusing device according to any one of claims 1 to 3, wherein the specimen is a well plate or a slide glass.   The image capturing apparatus including the autofocus device according to any one of claims 1 to 3, wherein the stage is an electric stage capable of freely moving the specimen on the XY plane.   The image capturing apparatus provided with the autofocus device according to any one of claims 1 to 3, wherein the image capturing means is a cooled CCD camera.   The image pickup device provided with the autofocus device according to any one of claims 1 to 3, wherein the optical element is an excitation filter, a dichroic mirror, and an absorption filter.   The pupil that irradiates the sample with laser light through the objective lens, receives the reflected light at the light receiving unit through the objective lens, and performs focusing based on the calculation result. The image pickup apparatus provided with the autofocus device according to any one of claims 1 to 3, wherein the image pickup apparatus is a division method.

Images