JP2006023491A - Microscopic imaging apparatus and biological material observation system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscopic imaging apparatus capable of accurately measuring an object to be inspected having drastic variance in luminance, and to provide a biological material observation system with which the microscopic imaging apparatus is used. <P>SOLUTION: The microscopic imaging apparatus is provided with a stage for holding the object to be inspected, an illuminating means for illuminating the object, an imaging means for photographing the image of the object, and a moving means for relatively moving the stage and the imaging means, and the imaging means is provided with an imager capable of imaging by a time-delay integration system, and in the case of imaging (S1 and S3) the object by the imaging means plural times so as to capture a plurality of sheet images, an exposure time for generating the accumulated charge in the imager is varied whenever capturing a different image, and the plurality of sheet images are synthesized (S4) to one sheet image. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microscope imaging apparatus used in a microscope and a biological sample observation system using the same.

  A device using a microplate is known as a device for measuring fluorescence of a biological sample. Since the fluorescence intensity of this biological sample is extremely dark, exposure to fluorescence for a long time is required. The microplate has an outer shape larger than the field of view of the microscope. Therefore, it takes time to observe the entire microplate.

A time-delay integration (TDI) method is known as an imaging method for observing a sample as described above for a long time and at a high speed (for example, see Patent Document 1).
Imaging by the TDI method is as follows. An image sensor used in the TDI system is composed of a plurality of photoelectric elements. Fluorescence from a biological sample is incident on this photoelectric element. In the photoelectric element, a charge corresponding to the fluorescence is generated by photoelectric conversion. Here, this electric charge is transferred in the photoelectric element corresponding to the movement of the microplate. Since the movement of the electric charge corresponds to the movement of the microplate, the fluorescence from the same location of the biological sample is incident again on the photoelectric element after the movement. As a result, charges are added. Thus, in the TDI system, charges are accumulated sequentially.
As described above, the feature of the TDI method is that the charges corresponding to the fluorescence are integrated while being transferred. Therefore, the stage can be moved earlier by the number of charge transfer lines compared to the case of imaging using a one-dimensional line sensor. As a result, the measurement time can be shortened.
International Publication No. 03/014400 (Fig. 1 etc.)

However, the technique described in Patent Document 1 described above has the following problems. For example, it is assumed that a sample having a large variation in brightness is measured, such as fluorescence observation of a biological sample. In this case, it is assumed that an image is captured with an exposure time suitable for an area with low fluorescence brightness (low brightness area). As a result, there is a risk that light and darkness in a region with high fluorescence luminance (high luminance region) cannot be detected.
In other words, the exposure time becomes a time that is not suitable (too long) for imaging in the high-luminance area, when it is adapted to shooting in the low-luminance area. Then, since the high brightness area is imaged with an inappropriate exposure time, the accumulated charge in the photoelectric element corresponding to the high brightness area is saturated. As a result, there is a possibility that light and darkness in the high luminance area cannot be detected.
Conversely, it is assumed that an image is captured with an exposure time suitable for capturing a high luminance area. In this case, the exposure time is shortened. Therefore, the accumulated charge in the photoelectric element corresponding to the low luminance region is not sufficiently accumulated. For this reason, there is a risk that the brightness of the low luminance area cannot be detected.

  The present invention has been made to solve the above-described problems, and is a microscope imaging apparatus capable of performing accurate measurement even on an object to be inspected with large variations in luminance, and biological sample observation using the same The purpose is to provide a system.

In order to achieve the above object, the present invention provides the following means.
According to a first aspect of the present invention, a stage for holding an object to be inspected, an illuminating means for illuminating the object to be inspected, an image capturing means for capturing an image of the object to be inspected, and the stage and the image capturing means are relative to each other. Moving means, and the imaging means generates an accumulated charge corresponding to the object to be inspected, and transfers the accumulated charge corresponding to the relative movement between the stage and the imaging means, When an imaging device capable of imaging by a time delay integration method that sequentially accumulates and accumulates the accumulated charges is provided, and the imaging unit captures the inspection object a plurality of times to acquire a plurality of images In addition, there is provided a microscope imaging apparatus for combining the plurality of images into one image by varying the exposure time for generating the accumulated charge in the imaging device every time a different image is acquired.

According to the present invention, it is possible to obtain a single image with a wide dynamic range by acquiring a plurality of images of the inspection object having different exposure times by the imaging unit and combining these images into one image. .
That is, imaging is performed with a short exposure time for a region where the luminance of the inspection object is high, and imaging is performed with a long exposure time for a region where the luminance of the inspection object is low. Then, by synthesizing the images obtained respectively, it is possible to obtain an image capable of measuring the brightness of the high brightness region and the brightness of the low brightness region.

Further, in the above invention, the apparatus further comprises an exposure time input means for inputting the exposure time from the outside, wherein the imaging means images the inspection object using the input exposure time, and the input exposure It is desirable to determine the length of the exposure time used for the next imaging based on the image acquired by imaging using the time.
More preferably, the exposure time input by the exposure time input means is set as the longest exposure time that is the upper limit of the exposure time.

According to the present invention, the length of the exposure time used for the next imaging is determined from the image of the inspection object based on the exposure time input from the outside. Therefore, an image with a wide dynamic range can be obtained efficiently.
Also, by setting the exposure time input from the outside as the longest exposure time, an image with a wide dynamic range can be obtained more efficiently.
That is, by inputting the exposure time from the outside, an image of the object to be inspected can be obtained without performing pre-scanning or the like. Therefore, since the measurement time is shortened, the image acquisition can be made efficient. Further, by setting the input exposure time as the longest exposure time, the exposure time can be limited. That is, it is possible to prevent the exposure time from becoming unnecessarily long. Therefore, the image acquisition can be made more efficient.

  Note that the exposure time input to the exposure time input means is preferably performed by, for example, a user who measures the object to be inspected. This is because the user can predict the longest exposure time suitable for the inspected object in advance. As a result, it is possible to further increase the efficiency of image acquisition. Here, the longest exposure time suitable for the object to be inspected means an exposure time having a minimum length necessary for measuring the lowest luminance portion of the object to be inspected. Note that the exposure time may be input using a microscope imaging device.

Furthermore, in the said invention, it is desirable for the said imaging means to provide the image pick-up element which has a smear suppression function.
According to the present invention, it is possible to prevent the occurrence of smear from a high-luminance portion of the object to be inspected by using an image sensor having a smear suppressing function.
Therefore, it is possible to prevent formation of a region difficult to measure due to smear. For example, it is possible to accurately measure the low-luminance portion by preventing smear from the high-luminance portion.
Moreover, the invention which concerns on Claim 5 is a biological sample observation system provided with the said microscope imaging device.
According to the present invention, when observing a biological sample that has been operated so as to emit fluorescence, the imaging time interval can be shortened, and therefore, a phenomenon that occurs only for a short period of time unique to the biological sample. And the accuracy of the observation result of the state of the biological sample can be improved.

According to the microscope imaging apparatus of the present invention, when acquiring a plurality of images of an object to be inspected, it is possible to capture images with different exposure times of the imaging means. These images are combined into a single image. By doing in this way, there exists an effect that an exact measurement can be performed also to a to-be-inspected object with a large variation in brightness.
According to the biological sample observation system of the present invention, since the imaging time interval can be shortened, it is possible to capture a phenomenon that occurs only for a short period of time unique to the biological sample, and observe the state of the biological sample. There is an effect that the accuracy of the result can be improved.

[First Embodiment]
Hereinafter, a first embodiment of a microscope imaging apparatus according to the present invention will be described with reference to FIGS.

First, an imaging operation by the TDI method will be described with reference to FIG.
FIGS. 1A, 1B, and 1C are diagrams illustrating a TDI imaging operation. FIGS. 2A, 2B, and 2C are diagrams for explaining signal charge transfer in the TDI system. 3A is a diagram showing the intensity of the signal charge accumulated in the horizontal line LA, and FIG. 3B is a diagram showing the intensity of the signal charge accumulated in the horizontal line LB. (C) is a figure which shows the intensity | strength of the signal charge accumulate | stored in the horizontal line LC.
Here, for ease of explanation, a star-shaped sample is used. However, the shape of the sample is not particularly limited to this shape.

  An arrangement relationship between the sample (inspection object) 20 and the imaging area S of the imaging element 63 is shown in FIG. In this way, imaging starts from a positional relationship in which the upper side of the sample 20 and the lower side of the imaging area S overlap. Therefore, first, only the tip region of the upward convex portion is projected onto the image sensor 63. At this time, an image of the tip region of the upward convex portion is taken by the horizontal line LA of the image sensor 63 as shown in FIG. As shown in FIG. 3A, signal charges having an intensity corresponding to the amount of light (brightness) from the sample 20 are accumulated in the horizontal line LA.

Next, the stage 30 is moved at a predetermined timing. Here, as shown in FIG. 1B, the image sensor 63 is moved by one horizontal line in the positive direction of the Y axis. Then, the whole upward convex part of the sample 20 is imaged.
At this time, as shown in FIG. 2B, simultaneously with the movement of the stage 30, the signal charge accumulated in the horizontal line LA is transferred to the horizontal line LB. Then, an image of the sample 20 is taken by the horizontal line LA and the horizontal line LB of the image sensor 63.
Therefore, as shown in FIG. 3B, the signal charge accumulated at the previous time and the signal charge accumulated at the current photographing are added to the horizontal line LB. That is, the signal charges obtained in the previous and current photographing are accumulated on the horizontal line LB. As a result, the signal charge is twice as strong as when measured on one line.
Thus, charge transfer is performed from the state shown in FIG. 2A to the state shown in FIG. The time interval at which this charge is transferred is called the TDI line transfer rate.

Further, the stage 30 is moved at a predetermined timing. That is, as shown in FIG. 1C, the image sensor 63 is moved by one horizontal line in the positive direction of the Y axis. Thereby, the imaging of the sample 20 is continued.
At this time, as shown in FIG. 2C, simultaneously with the movement of the stage 30, the signal charges accumulated in the horizontal line LB are transferred to the horizontal line LC. Further, the signal charges accumulated in the horizontal line LA are transferred to the horizontal line LB. Then, an image of the sample 20 is taken by the horizontal line LA, the horizontal line LB, and the horizontal line LC.
Therefore, as shown in FIG. 3C, the signal charge obtained at the time of the current photographing is added to the horizontal line LC. Therefore, the signal charges obtained in the previous and current shootings are accumulated in the horizontal line LC two times in advance. That is, the signal charge is three times stronger than when measured on one line. In the horizontal line LB, signal charges obtained at the previous and current photographing are accumulated.

  By continuing the above operation, photographing is performed as many times as the number of horizontal scanning lines. As a result, signal charges corresponding to the same part of the sample 20 are accumulated as many times as the number of photographing. As described above, in the TDI system, the image of the sample 20 projected on the imaging surface shifts with the movement of the stage 30, but the signal charges accumulated in the imaging device 63 are shifted in synchronization with the movement.

Next, the exposure time at the time of shooting in the TDI system will be described.
The exposure time in the TDI system is a time during which the signal light returned from the sample 20 is accumulated as a signal charge. That is, the exposure time in the TDI system can be expressed as a product of the TDI line transfer rate and the cumulative number of pixels. Therefore, to change the exposure time, the exposure time can be shortened by increasing the exposure time by increasing the TDI line transfer rate, or by decreasing the line transfer rate.
For example, assume that a CCD camera having 1000 pixels in the Y-axis direction is used as the image sensor 63. In this case, in order to capture an image with an exposure time of 0.2 (s), the transfer rate may be set to 5 kHz (0.2 ms) by the following calculation.
0.2 (s) /1000=0.2 (ms)

Next, the stage scanning speed in the TDI system will be described.
The movement of the stage 30 is performed in synchronization with the transfer of signal charges. Therefore, the moving speed of the stage 30 can be represented by a value obtained by dividing the pixel size on the stage 30 by the TDI line transfer rate. Here, the pixel size on the stage 30 can be obtained from the pixel size of the image sensor 63 (for example, CCD) and the projection magnification.
For example, the projection magnification is 10 times, the pixel size of the image sensor 63 is 6.45 (μm), and the TDI line transfer rate is 5 kHz. The stage speed at this time is 3.23 (mm / s) according to the calculation formula shown below.
6.45 × 10 ⁻³ (mm) / 10 × 5 × 10 ³ (Hz) = 3.23 (mm / s)

Next, the configuration of the microscope imaging apparatus in the present embodiment will be described.
FIG. 4 is a diagram illustrating an overall configuration of the microscope imaging apparatus according to the present embodiment.
As shown in FIG. 4, the microscope imaging apparatus 10 is schematically configured by a stage 30, an illumination device (illuminating means) 40, and an imaging device (imaging means) 60. The stage 30 holds the sample (inspection object) 20 and is movable. The illumination device 40 irradiates the sample 20 with illumination. The imaging device 60 captures and measures signal light generated from an illumination area.

  The lighting device 40 includes a component capable of Koehler illumination. Specifically, the lamp 41, the collector lens 42, the reflecting mirror 43, the field stop 44, and the lens 45. A gas discharge lamp or the like is used as the light source lamp 41. Examples of the gas discharge lamp include a halogen lamp, a xenon lamp, and a mercury lamp. The collector lens 42 collects light emitted from the lamp 41. The reflecting mirror 43 reflects light emitted backward from the lamp 41 back to the lamp 41. A field stop 44 is disposed behind the collector lens 42. The field stop 44 is arranged at a conjugate position with a focal plane of an objective lens 49 described later. In addition, the field stop 44 has an adjustable opening diameter. A lens 45 is disposed behind the field stop 44. The lens 45 images the field stop 44 at infinity.

An aperture stop 46 is disposed on the focal plane of the lens 45 behind the lens 45. The aperture stop 45 can change the diameter of the aperture. Thereby, the size of the light beam diameter at the exit pupil position of the objective lens 49 is adjusted. A mirror 47 is disposed behind the aperture stop 46. As the mirror 47, a dichroic mirror that reflects light from the lamp 41 and transmits return light (fluorescence) from the sample 20 is used. A half mirror may be used instead of the dichroic mirror.
An objective lens 49 is disposed behind the mirror 47. A stage 30 is disposed at a position facing the objective lens 49.

  A stage driving mechanism (moving means) 31 is disposed on the stage 30. The stage drive mechanism (moving means) 31 controls the drive of the stage 30 in the XY directions based on the output of a computer to be described later. As the stage drive mechanism 31, a known technique, for example, a slide movement mechanism can be used. However, it is not particularly limited to the slide movement mechanism.

In the imaging device 60, an imaging lens 61 and a detector 62 are arranged. Return light (fluorescence) from the sample 20 enters the imaging lens 61. The imaging lens 61 condenses (images) incident light at a predetermined position. The detector 62 is disposed at a predetermined position. The detector 62 detects return light from the sample 20. The detector 62 is provided with an image sensor 63 capable of imaging by the TDI method. As described above, the image sensor 63 accumulates signal charges accumulated in each horizontal line in the element in accordance with the movement of the stage 30.
The output of the detector 62 is connected to an image processing unit 64 that processes the output signal from the detector 62. A monitor 65 for displaying the processed signal is connected to the image processing unit 64. The image processing unit 64 is connected to the computer 66. The stage drive mechanism 31 is also connected to the computer 66.
The computer (calculation means) 66 calculates the exposure time in the main measurement. As will be described later, this calculation is performed based on information acquired in the pre-scan. This information is the luminance in each specimen arrangement unit.

FIG. 5A illustrates the shape of the sample 20. FIG. 5B is an enlarged view showing the specimen arrangement part 22 of the sample 20.
The sample 20 has a transparent substrate 21 as shown in FIG. The transparent substrate 21 is made of a material such as a glass plate or a plastic plate. The transparent substrate 21 has sample arrangement portions 22 formed in a matrix. That is, the two-dimensional pattern portion 23 is formed by the plurality of specimen arrangement portions 22.
The specimen arrangement portion 22 is formed in a substantially circular shape having a diameter of about several millimeters in a plan view and a concave shape in a cross-sectional view. And in the sample arrangement | positioning part 22, as shown in FIG.5 (b), the cell which is a measuring object is arrange | positioned. Further, the cells are cultured in the specimen arrangement unit 22.

Next, stage scanning in the TDI system will be described.
FIG. 6 is a diagram for explaining scanning in the TDI system. In this embodiment, the stage 30 is moved for scanning. However, in FIG. 6, for ease of illustration, the image pickup device 63 is described as being moved.

When the sample 20 is measured by the TDI method, the entire surface of the two-dimensional pattern unit 23 is scanned as shown in FIG. This scanning is performed regardless of the presence / absence of the sample placement unit 22.
Here, when the stage 30 is scanned in the Y-axis direction, the stage 30 moves at a constant speed in the -Y-axis direction. On the other hand, the image sensor 63 can transfer charges only in one direction. Therefore, as shown in FIG. 6, the measurement is performed only when the stage 30 advances in the −Y axis direction. Note that no measurement is performed when the stage 30 advances in the + Y-axis direction. The movement in the + Y-axis direction is a movement for returning the stage to the initial position. The time until returning to the initial position can be applied to the time for recording the acquired data on the hard disk of the computer 66, for example.

Next, the measurement procedure will be described with reference to FIG.
FIG. 7 is a flowchart showing a measurement procedure in the present embodiment. FIG. 8 is a diagram illustrating the correlation between the luminance of the image of the sample 20 and the output level from the image sensor. Here, FIG. 8A is a diagram showing the correlation between the luminance and the output level acquired in the first scan. FIG. 8B is a diagram showing the correlation between the luminance acquired in the second scan and the output level. FIG. 8C is a diagram showing the correlation between the luminance and the output level acquired in the first and second scans.

First, the first scanning (imaging) is performed to acquire an image of the sample 20 (S1). This corresponds to pre-scanning. Here, the sample 20 is imaged with a predetermined exposure time while moving the sample 20 (stage 30) from the position a in FIG. 6 to the position b.
For the image acquired in the first scan, the correlation between the brightness of the image and the output level from the image sensor 63 is examined. In this case, for example, as shown in FIG. 8A, there are a portion where the accumulated charge is saturated (hereinafter referred to as a saturation level portion) and a portion where the accumulated charge is very small (hereinafter referred to as a trace level portion). Exists.

Next, preparation for the second scan is performed (S2).
Here, regarding the saturation level portion and the minute level portion, the output levels of these portions are made appropriate. That is, an exposure time having an appropriate length is set for each of these portions.
That is, when eliminating the saturation level portion, the length of the exposure time is set shorter than the exposure time in the first scan. At this time, the output level is set to a level where the dynamic range of the image sensor 63 is used up. Further, when eliminating the minute level portion, the length of the exposure time is set longer than the exposure time in the first scanning. Also in this case, the output level is set to a level where the dynamic range of the image sensor 63 is used up.
At the same time, the position of the sample 20 (stage 30) is moved from b to a.
In the present embodiment, the description will be made with reference to an example of eliminating the saturation level portion.

Next, the second scanning (imaging) is performed, and an image of the sample 20 is acquired again (S3).
In this imaging, the sample 20 is imaged in the same manner as S1 using the exposure time obtained in S2.
It can be said that the image acquired by the second scanning shows a correlation between the brightness of the obtained image and the output level from the image sensor 63. FIG. 8B shows an image acquired by the second scan. As shown in the figure, the saturation state is eliminated at the saturation level portion in the previous scan. On the other hand, the output level is increased at the minute level portion.

Next, the images acquired in the first and second scans are combined into one image (S4).
Here, the images acquired in S1 and S3 are combined. Thereby, one image with a wide dynamic range is obtained.
Thus, in the present embodiment, as shown in FIG. 8C, one image is obtained using the image acquired in S1 with a long exposure time and the image acquired in S3 with a short exposure time. The image is synthesized.
Thereafter, the sample 20 (stage 30) is moved from the position b to the position c, and the measurement preparation for the next line is started.

  In the present embodiment, an example in which scanning at the same location is repeated twice is described. However, the number of scans is not limited to two. For example, the scanning of the same portion may be repeated three times or more times. In this case, preparation for the next scan is performed during the acquisition of the image by each scan, and the synthesis of the acquired image is performed last.

In addition to the image sensor 63 described above, an image sensor 63a having an electronic shutter may be used. Use of the image sensor 63a is preferable because the exposure time can be shortened.
The electronic shutter is a shutter used in a digital camera using a CCD or a mobile phone with a camera. This electronic shutter utilizes a phenomenon that charges are not accumulated if the CCD is not operating even when exposed to light. Therefore, the CCD itself is a kind of shutter.
On the other hand, a mechanical shutter used in a silver halide camera places a plate that blocks light between a lens and a film, and performs exposure by opening and closing (moving) the plate. Therefore, the electronic shutter is different from the mechanical shutter. Thus, in the electronic shutter, the exposure time is controlled without using a mechanical shutter.

FIG. 9 is a time chart of the electronic shutter in the TDI system.
For example, as shown in FIG. 9, assuming that the fastest transfer rate in the TDI system is t1 seconds, charges are transferred to the adjacent horizontal line every t1 seconds. The transfer timing is indicated by an arrow in the figure.
The electronic shutter is open for t2 seconds from each charge transfer timing, and charges are accumulated. That is, charge accumulation of the CCD is t2 seconds per charge transfer timing. On the other hand, the exposure time when the electronic shutter is not used is t1. Therefore, the exposure time when the electronic shutter is used is t2 / t1 times that when the electronic shutter is not used.
For example, it is assumed that the imaging device 63a is an imaging device having 1000 pixels in the Y-axis direction and a fastest transfer rate (t1) of 10 kHz (0.1 ms). In order to capture an image with an exposure time of 1 ms using the image sensor 63a, the electronic shutter Open time (t2) may be set to 0.001 ms by the following calculation.
1000 × 0.1 (ms) × (t2 / 0.1) = 1 (ms)
t2 = 0.001 (ms)
If the electronic shutter is not used, the shortest exposure time is determined by the product of the TDI fastest transfer rate and the number of integrated pixels. On the other hand, the measurable exposure time range can be expanded by using the electronic shutter as described above. Therefore, the high-intensity sample 20 can also be accurately measured.

  According to the above configuration, two images obtained by the two scans S1 and S3 having different exposure times are combined into one image (S4). By doing in this way, an image with a wide dynamic range can be obtained compared with the image obtained by one exposure time. Therefore, accurate measurement can be performed even for the sample 20 having a large variation in luminance.

As shown in FIG. 5, the sample 20 may have only the specimen arrangement portion 22 formed on the transparent substrate 21. Alternatively, as shown in FIG. 10, the reference line BL may be formed in a region near the left end of the transparent substrate 21.
When the reference line BL is formed, the reference line BL can be used as a reference when the images acquired by the respective scans are combined. Therefore, it is possible to facilitate image composition.
In the present embodiment, since fluorescence observation is performed, the reference line BL is preferably formed of a material that emits fluorescence.

[Second Embodiment]
Next, a second embodiment of the present invention will be described with reference to FIGS.
The basic configuration of the microscope imaging apparatus of the present embodiment is the same as that of the first embodiment. However, the sample measurement method is different from the first embodiment. Therefore, in this embodiment, only the sample measurement method will be described with reference to FIGS. 11 to 13 and description of the TDI method and the like will be omitted.
FIG. 11 is a diagram illustrating an overall configuration of the microscope imaging apparatus according to the present embodiment.

  As shown in FIG. 11, the microscope imaging device 110 is schematically configured by a stage 30, an illumination device 40, and an image device (imaging unit) 160. The stage 30 holds the sample 20 and is movable. The illumination device 40 irradiates the sample 20 with illumination light. The image device 160 captures and measures signal light generated from an illumination area.

In the imaging device 160, an imaging lens 61 and a detector 62 are arranged. Return light from the sample 20 enters the imaging lens 61. The detector 62 detects return light from the sample 20.
The detector 62 is connected to an image processing unit 64 that processes the output of the detector 62. The image processing unit 64, the monitor 65 that displays the processed signal, and the stage driving mechanism 31 are connected to the computer 166.
The computer 166 is provided with an exposure time input unit (exposure time input means) 167 and a determination unit 168. The exposure time input unit 167 receives the longest exposure time Tmax. The longest exposure time Tmax is a time related to the measurement of the sample 20, and is set by the user. Further, the determination unit 168 determines an exposure time Tmeasure used for photographing and measurement.

Next, the measurement procedure will be described.
FIG. 12 is a flowchart showing a measurement procedure in the present embodiment.
First, the user sets the longest exposure time Tmax (S10). The set longest exposure time Tmax is input to the exposure time input unit 167.
Next, the first scanning (imaging) is performed to obtain an image of the sample 20 (S11).
Here, the longest exposure time Tmax input to the determination unit 168 is set as the exposure time Tmeasure used for photographing and measurement. Therefore, an image of the sample 20 is acquired with the longest exposure time Tmax.

Next, preparation for the second scan is performed (S12).
Here, the determination unit 168 determines the exposure time Tmeasure in the second scan from the size of the saturation level portion (the number of saturated pixels).
Specifically, as shown in FIG. 13, the determination unit 168 stores a correlation (table) between the number of saturated pixels and the exposure time in the next imaging. Therefore, based on this table, the computer determines the exposure time Tmeasure in the second scan. Therefore, for example, when the number of saturated pixels per specimen arrangement unit 22 increases, the second exposure time is shortened.

A saturated pixel will be described. For example, assume that a 12-bit CCD is used as the image sensor 63. In this case, the maximum gradation is 4095 (= 2 ¹² : the maximum value of 12 bits). Therefore, the saturated pixel is a pixel when the image sensor outputs a value that is 4095. In other words, the pixel whose output value has reached the upper limit.

Next, the second scanning (imaging) is performed to obtain an image of the sample 20 (S13), and the images obtained by the first and second scannings are combined into one image (S14).
Since the measurement procedure after S13 is the same as that in the first embodiment, FIG. 12 is shown and the description thereof is omitted.

  According to the above configuration, the user inputs the longest exposure time Tmax to the exposure time input unit 167 in advance. By doing in this way, how to change exposure time can be limited to the direction to shorten. Therefore, the image acquisition can be made more efficient. Therefore, an image with a wide dynamic range can be acquired efficiently.

  In the present embodiment, an example is described in which the user sets the longest exposure time. However, the time input to the exposure time input unit 167 may be a simple exposure time. That is, it is not limited to the longest exposure time.

[Third Embodiment]
Next, a third embodiment of the present invention will be described with reference to FIGS.
The basic configuration of the microscope imaging apparatus of the present embodiment is the same as that of the first embodiment. However, the configuration of the imaging device is different from that of the first embodiment. Therefore, in this embodiment, only the periphery of the imaging device will be described with reference to FIGS. 14 and 15, and the description of the illumination device and the like will be omitted.
FIG. 14 is a diagram illustrating an overall configuration of the microscope imaging apparatus according to the present embodiment.
As shown in FIG. 12, the microscope image pickup device 210 is schematically configured by a stage 30, an illumination device 40, and an image pickup device (image pickup means) 260. The stage 30 holds the sample 20 and is movable. The illumination device 40 irradiates the sample 20 with illumination light, and the imaging device 260 captures and measures signal light generated from the region irradiated with the illumination light.

  The imaging device 260 is provided with an imaging lens 61 and a detector 262. Return light from the sample 20 enters the imaging lens 61. The detector 262 detects return light from the sample 20. An image sensor 263 is disposed in the detector 262. The image sensor 263 has a smear suppressing function and can capture images by the TDI method. As described above, the TDI method is a method of accumulating signal charges accumulated in each horizontal line in the element in accordance with the movement of the stage 30.

Here, smear is a phenomenon that occurs in a photoelectric conversion element such as a CCD (Charge Coupled Device). This phenomenon is that when an excess signal of a certain level or more is input to the photoelectric conversion element, the signal in the vertical or horizontal direction changes as a whole in the signal processing process to generate a pseudo signal.
That is, for example, when high-intensity spot light enters the photoelectric conversion element, a bright band extending in the vertical or horizontal direction is generated.

According to said structure, generation | occurrence | production of this smear can be prevented. For example, as shown in FIG. 15, it is assumed that a cell BC having a high luminance and a cell DC having a low luminance are adjacent to each other. In this case, smear occurs in the image of the cell BC with high luminance when exposed to the cell (biological sample) DC with low luminance for a long time. However, according to the above configuration, it is possible to prevent smear even when imaging is performed with a long exposure time in accordance with the low-brightness cell DC. Therefore, both the high brightness cell BC and the low brightness cell DC can be accurately measured.
In addition, when an image sensor that does not have a smear suppressing function is used, smear due to a cell BC having a high luminance may occur on the cell DC having a low luminance as described above. As a result, there is a possibility that the low-brightness cell DC cannot be measured accurately.

[Fourth Embodiment]
Next, a fourth embodiment of the present invention will be described with reference to FIGS. The fourth embodiment is a biological sample observation system to which any one of the above-described microscope imaging apparatuses according to the first to third embodiments is applied.
FIG. 16 is a perspective view showing an outline of the biological sample observation system according to the present embodiment. FIG. 17 is a schematic diagram showing the system configuration of the biological sample observation system.

  The biological sample observation system 410 is generally configured by a detection unit 420 and a culture unit 470, as shown in FIGS. The detection unit 420 and the culture unit 470 are desirably arranged close to each other, and more preferably, both the units 420 and 470 are disposed in contact with each other.

As shown in FIGS. 16 and 17, the detection unit 420 is roughly configured by a heat insulating box 421 that houses a biological sample therein, and a detection unit (microscope imaging device) 440 that measures a cell CE as the biological sample. Yes.
The heat insulation box 421 includes a heater 421H that keeps the inside of the heat insulation box 421 at a predetermined temperature, a stage 422 that holds an incubator box 500 described later, a transmission light source (illuminating means) 423 that irradiates light to the cells CE, and heat insulation. A fan 424 that makes the temperature in the box 421 uniform, a UV lamp 425 that sterilizes the inside of the heat insulation box 421, a carrier 426 that protects a culture solution circulation pipe 477 and a culture gas supply pipe 497, which will be described later, an incubator box 500, and the like Is provided with an open / close door 427 used when taking out and putting in and out of the heat insulation box 421 and a main power switch 428 for turning on / off the main power of the detection unit 420.

The stage 422 includes an X-axis operation stage (stage) 422X and a Y-axis operation stage (stage) 422Y that move relative to each other in an orthogonal direction, and scanning control is performed by a stage scanning unit (movement means) 429.
The stage scanning unit 429 includes an X-axis coordinate detection unit 430 that detects an X-axis coordinate value of the X-axis operation stage 422X, an X-axis scan control unit 431 that controls the operation (scanning) of the X-axis operation stage 422X, and a Y-axis A Y-axis coordinate detection unit 432 that detects the Y-axis coordinate value of the operation stage 422Y, and a Y-axis scan control unit 433 that controls the operation (scanning) of the Y-axis operation stage 422Y.
The X-axis coordinate detection unit 430 and the Y-axis coordinate detection unit 432 are arranged to output the detected X-coordinate of the X-axis operation stage 422X and Y-coordinate of the Y-axis operation stage 422Y to the computer PC. . The X-axis scanning control unit 431 and the Y-axis scanning control unit 433 are arranged so as to control scanning of the X-axis operation stage 422X and scanning of the Y-axis operation stage 422Y based on instructions from the computer PC.

As a mechanism for driving the X-axis operation stage 422X and the Y-axis operation stage 422Y, for example, a combination of a motor and a ball screw can be used.
As described above, the computer PC controls the scanning of the X-axis operation stage 422X and the Y-axis operation stage 422Y, controls the detection system of the cell CE, and analyzes the captured image of the cell CE as described later. The X-axis operation stage 422X, the Y-axis operation stage 422Y, the detection system, and the analysis system are controlled in conjunction with each other.

A condenser lens 434 that condenses the light emitted from the transmissive light source 423 onto the cell CE is disposed between the transmissive light source 423 and the incubator box 500.
Note that the shutter 435 may not be provided between the condenser lens 434 and the incubator box 500, or the shutter 435 may be provided.
The fan 424 is disposed on the wall surface of the heat insulating box 421. By operating the fan 424, the air in the heat insulation box 421 can be convected, and the temperature in the heat insulation box 421 can be easily maintained uniformly.

The UV lamp 425 is connected to a UV lamp switch 436 disposed on the wall surface of the detection unit 420, and a timer 437 that temporally controls the operation of the UV lamp 425 is provided between the UV lamp 425 and the UV lamp switch 436. Is arranged. Further, a sterilization indicator lamp (not shown) for displaying the lighting of the UV lamp 425 is arranged.
For example, when the UV lamp switch 436 is pressed when the cell CE is not measured, the timer 437 starts counting, power is supplied to the UV lamp 425, and UV light (ultraviolet light) is irradiated into the heat insulating box 421. The At the same time, the sterilization indicator lamp is lit. When a predetermined time (for example, 30 minutes) elapses and the timer 437 finishes counting, the timer 437 stops supplying power to the UV lamp 425, and the UV light irradiation ends. The sterilization indicator lamp is also turned off.
The UV lamp 425 is controlled separately from the main power switch 428 and can operate even when the main power is turned off.
The lighting time of the UV lamp 425 may be 30 minutes as described above, or may be less than 30 minutes or longer than 30 minutes as long as it can kill the germs in the heat insulation box 421. Also good.

The opening / closing door 427 is made of an alumite-treated metal such as aluminum, or a translucent resin having a high light shielding property.
As the structure of the opening / closing door 427, a structure having a hollow double structure, or a structure in which the inside is made of the above metal and the outside is made of resin can be considered. By using resin on the outside of the opening / closing door 427, heat in the heat insulating box 421 can be prevented from escaping from the opening / closing door 427 to the outside. In addition, by making the inside of the opening / closing door 427 an anodized metal, it is possible to prevent the life of the opening / closing door 427 from being impaired by the UV lamp 425.
Note that, when the opening / closing door 427 has a double structure of metal or metal and resin, since the light is completely shielded, it is desirable to provide a viewing window at a position where the incubator box 500 is viewed. It is desirable that a transparent resin or glass is fitted in the viewing window, and a cover that can be opened and closed is disposed on the outside.

  As shown in FIGS. 16 and 17, the detection unit 440 includes a heater 440H that keeps the inside of the detection unit 440 at a predetermined temperature, epi-illumination light sources 441A and 441B that irradiate cells CE from the detection unit 440 side, and an epi-illumination light source. An optical path switching unit 442 that switches the optical paths from 441A and 441B, a light amount adjustment mechanism 443 that adjusts the amount of irradiation light, a lens system 444 that collects the irradiation light toward the cell CE, and the wavelength and detection light of the irradiation light A filter unit 445 for controlling the wavelength of the light, an autofocus (AF) unit 446 for performing a focusing operation on the cell CE, a revolver 447 including a plurality of objective lenses 448 having different magnifications and properties, and a cell CE A detector (imaging means) 449 for detecting the detection light, a light amount monitor 450 for measuring the light amount of the detection light, and the temperature in the detection unit 440 are made uniform. A fan 451, a cooling fan 452 for cooling the inside detection part 440, is provided.

The epi-illumination light sources (illuminating means) 441A and 441B are composed of, for example, mercury lamps, and are disposed outside the detection unit 440 and connected to a power source 453 that supplies power.
Normally, one incident light source, for example, the incident light source 441A is used. However, when the amount of light from the incident light source 441A decreases to a predetermined value or less, illumination light is emitted from the incident light source 441B, and the power source of the incident light source 441A is supplied. Is turned off.

The optical path switching unit 442 is formed to guide the illumination light of either the incident light source 441A or the incident light source 441B to the light amount adjusting mechanism 443. The optical path switching unit 442 is provided with an optical path control unit 454 that is connected to a computer PC described later and controls the optical path switching unit 442 based on an instruction from the computer PC.
A shutter 442S is disposed on the illumination light exit side of the optical path switching unit 442, and performs transmission / blocking control of illumination light.

A light amount adjusting mechanism 443 is disposed on the illumination light emission side of the shutter 442S to adjust the amount of illumination light transmitted through the shutter 442S. As the mechanism, for example, a known diaphragm mechanism or the like may be used, or a known mechanism or technique capable of adjusting the other light amount may be used.
The light amount adjustment mechanism 443 is provided with a light amount control unit 455 that is connected to a computer PC described later and controls the light amount adjustment mechanism 443 based on an instruction from the computer PC.
A lens system 444 is disposed on the illumination light exit side of the light amount adjustment mechanism 443. The lens system 444 includes a pair of lenses 444A and 444B, and a stop 444C disposed between the lens 444A and the lens 444B.

The filter unit 445 includes an excitation filter 456, a dichroic mirror 457, and an absorption filter 458. The excitation filter 456 is a filter that transmits the wavelength (excitation light) that contributes to the fluorescence emission of the cell CE from the illumination light, and is arranged so that the illumination light emitted from the lens system 444 enters the excitation filter 456. ing. The dichroic mirror 457 is an optical element that separates excitation light and fluorescence. The dichroic mirror 457 is arranged so as to reflect the excitation light transmitted through the excitation filter 456 toward the cell CE and to transmit fluorescence from the cell CE. Yes. The absorption filter 458 is an optical element that separates fluorescence from the cell CE and other unnecessary scattered light, and is arranged so that light transmitted through the dichroic mirror 457 enters.
The filter unit 445 is provided with a filter control unit 446C that controls the wavelengths of excitation light and detection light (fluorescence) emitted from the filter unit 445 based on instructions from the computer PC, which will be described later.
In addition, the excitation filter 456, the dichroic mirror 457, and the absorption filter 458 may be used one by one, or a plurality may be used.

The AF unit 446 is arranged on the excitation light emission side of the filter unit 445, and is arranged so as to collect the excitation light on the cell CE via the objective lens 448 based on an instruction from the computer PC described later.
The revolver 447 is disposed on the excitation light emission light side of the AF unit 446, and a plurality of objective lenses 448 having different magnifications are disposed. The revolver 447 is provided with an objective lens control unit 459 that selects and controls the objective lens 448 on which the excitation light is incident, based on an instruction from the computer PC described later.

The objective lens 448 has a structure in which the inside of the incubator box in the heat insulating box 421 can be observed through the holes provided in the X-axis operation stage 422X and the Y-axis operation stage 422Y from the detection unit 440.
In the X-axis operation stage 422X and the Y-axis operation stage 422Y, the size of the hole is expected to be somewhat marginal in consideration of the range in which the stage operates.
Therefore, even if the atmosphere is kept at a humidity suitable for cell culture in the heat insulating box 421, the atmosphere escapes to the detection unit 440 through the hole, and the temperature suitable for cell culture cannot be maintained. May cause decreased activity.
Therefore, suppression means 499 may be provided that suppresses conduction of an atmosphere having a temperature suitable for such cell culture between the heat insulating box 421 and the detection unit 440.
The suppression unit 499 only needs to be able to suppress the flow of air so as not to interfere with the movement of the revolver 447 and the objective lens 448. It is possible to use a curtain-like one that is attached around a hole provided in the slab and attached in a suspended state around the revolver.

A condensing lens 460 that condenses the detection light on the detector 449 and the light amount monitor 450 is disposed on the detection light emission side of the filter unit 445.
A half mirror 461 that reflects part of the detection light toward the detector 449 and transmits the remaining detection light toward the light amount monitor 450 is disposed on the detection light emission side of the condenser lens 460.
The detector 449 is disposed at a position where the detection light reflected from the half mirror 461 is incident. The detector 449 is connected to a detector calculation unit 462 that calculates a detection signal from the detector 449 and outputs the detection signal to a computer PC described later.
Note that the detector 449 may use a line sensor, an area sensor, or may share the line sensor and the area sensor, and is not particularly limited.
The light amount monitor 450 is arranged to measure the detection light transmitted through the half mirror 461 and output the measured value to the computer PC.
As described above, the light amount of the detection light may be measured using the light amount monitor 450, or the light amount of the detection light may be measured using an illuminance meter, a power meter, or the like.

The heater 440H controls the temperature of the inside of the detection unit 440 so that the temperature becomes, for example, 30 ° C. to 37 ° C. The fan 451 is disposed so as to convect the air in the detection unit 440 and make the temperature in the detection unit 440 uniform. Therefore, the temperature in the detection unit 440 is maintained at a temperature close to that of the heat insulation box 421, and the temperature of the heat insulation box 421 is more easily stabilized.
The cooling fan 452 is driven to lower the temperature in the detection unit 440 based on the output of a temperature sensor (not shown) disposed in the detection unit 440. Therefore, for example, an abnormal temperature rise in the detection unit 440 due to heat generation of a motor or the like can be prevented.

FIG. 18 is a perspective view showing an incubator box according to the present embodiment, and FIG. 19 is a cross-sectional view of the chamber according to the present embodiment.
As shown in FIGS. 18 and 19, the incubator box 500 is generally configured from a chamber (inspection object) 510, a housing 501 for housing, and a cover 502 that forms a sealed space together with the housing 501. The housing 501 and the cover 502 are subjected to a magnetic shielding process for blocking a magnetic field from the outside and an electrostatic removal process for removing static electricity generated in the incubator box 500.

The housing 501 is formed of a bottom plate 503 and a side wall 504, and a region corresponding to the measurement area of the bottom plate 503 is formed of a light-transmitting material such as glass. The other region of the bottom plate 503 and the side wall 504 are preferably made of a light-shielding material having high anticorrosion properties such as anodized aluminum or stainless steel such as SUS316, and more preferably from the viewpoint of heat retention. A material with low thermal conductivity may be selected.
In addition, an adapter 505 for holding the chamber 510 and a temperature sensor 506 for measuring the temperature of the chamber 510 are arranged on the bottom plate 503. Note that the chamber 510 may be held using the adapter 505 as described above, or the chamber 510 may be held without using the adapter 505.
The output of the temperature sensor 506 is input to the computer PC via the incubator temperature detection unit 506S, and is also input to the temperature display unit 507 disposed on the wall surface of the detection unit 420. The computer PC controls the heater 421H and the like via an incubator temperature control unit 506C shown in FIG. 17 so that the temperature in the incubator box 500 is kept constant.

The cover 502 includes a glass plate 517 that transmits illumination light, and a support portion 517A that supports the glass plate 517. On the glass plate 517, antireflection films may be formed on both sides in a region corresponding to the measurement area. By forming antireflection films on both sides, reflection by the glass plate 517 in transmission observation and epi-illumination observation is prevented.
The area of the glass plate 517 may be substantially the same as that of the bottom plate 503 of the incubator box 500, or may be a minimum area necessary for performing measurement without any problem.

As shown in FIG. 19, the chamber 510 includes a lower glass member 511 for observing with an objective lens 448, an upper glass member 512 for transmitting light from the transmission light source 423, a lower glass member 511, and an upper glass member. It is roughly formed from a frame member 513 that supports 512.
On opposite sides of the frame member 513, a joint 514 in which a channel for circulating the culture solution is formed is formed. The joint 514 is connected to a culture solution circulation pipe 477 described later, so that the culture solution is circulated with the culture unit 470.
The frame member 513 is provided with a pair of commutators 515 that make the flow of the culture solution uniform, substantially perpendicular to the flow of the culture solution. The commutator 515 is made of, for example, a plate member in which small holes are formed in a matrix, and the flow is made uniform by flowing through a plurality of small holes formed by dispersing the culture medium. Between the two commutators 515, a slide glass 516 seeded with cells CE is disposed.

As described above, the incubator box 500 may have a chamber 510 disposed therein, or a microplate (inspection object) 520 (or well plate) may be disposed inside as shown in FIG. It may be arranged.
In the case of this configuration, as shown in FIG. 20A, the casing 501 of the incubator box 500a has a water tank 521 that surrounds the microplate 520 in a mouth shape, and an inner part disposed inside the water tank 521 A fan 522, a connector 523 for supplying culture gas, and a culture gas concentration sensor 524 for detecting the carbon dioxide gas concentration in the culture gas are arranged.
The temperature sensor 506 is arranged to measure the temperature of the microplate 520. The microplate temperature input from the temperature sensor 506 to the computer PC is accumulated as text data in the memory and can be processed in the computer PC.
The culture gas concentration sensor 524 outputs the carbon dioxide gas concentration to the computer PC and the culture gas concentration display unit 524D.

The side wall 521W of the water tank 521 is formed lower than the height of the side wall of the housing 501. Further, the arrangement relationship with the connector 523 is adjusted so that the supplied culture gas hits the side wall 521W. Sterilized water is stored in the water tank 521, and the humidity in the incubator box 500a is adjusted to approximately 100%.
The internal fan 522 is disposed so as to blow along the side wall 521W of the water tank 521 so that the microplate 520 is not disposed in the blowing direction.
The culture gas concentration sensor 524 may be arranged on the inner surface of the side wall 521W of the water tank 521, or a pipe is arranged outside from the incubator box 500a, and the culture gas in the incubator box 500a is sucked by a suction pump and cultured. The concentration may be detected by the gas concentration sensor 524.

  When such an incubator box 500a is used, it is necessary to change the culture gas supply destination of the culture gas mixing tank 491 described later from the culture solution bottle 472 to the incubator box 500a and to supply the culture solution from the culture unit 470. Therefore, the pumps such as the culture medium pump 480 are stopped.

By adopting such a configuration, the incubator box 500a maintains the humidity environment and the culture gas concentration in which the change or non-uniformity is less damaging to the cell CE compared to the temperature environment. Can be reduced.
Further, since the humidity and the culture gas concentration are maintained in the incubator box 500a that is not in direct contact with the detection unit 440, contamination during observation can be prevented.
Furthermore, since it is not necessary to maintain the humidity and culture gas concentration suitable for cell CE culture in the heat insulation box 421, the objective lens 448 and the like disposed in the heat insulation box 421 prevent the function from being impaired by the humidity. be able to. In addition, shortening of the life of the objective lens 448 and the like can be prevented.

As described above, the chamber 510 may be sealed, or may be an open chamber that is not sealed. The open chamber is formed in the same configuration as the chamber 510 except that the upper glass member 512 is not provided.
When an open chamber is used, the incubator box 500a described above is used to fill the incubator box 500a with a culture gas, and a culture solution is supplied to the open chamber.

Note that the chamber 510 described above may be used for the incubator box 500a as shown in FIG. In this case, the connector 523 for supplying the culture gas is closed and the culture gas concentration sensor 524 is not used. When the size of the chamber 510 is different from that of the microplate 520, the chamber 510 may be disposed in the incubator box 500a using the adapter 505. Moreover, it is not necessary to put sterilized water into the water tank 521, and the water tank 521 itself may be removed from the incubator box 500a. The temperature sensor 506 measures the temperature of the chamber 510.
The connector 523 may be closed as described above, or the supply of the culture gas to the incubator box 500a may be stopped while the culture gas supply pipe 497 is connected.

As shown in FIGS. 16 and 17, the culture unit 470 is generally configured by a sterilization box 471 that stores a culture solution therein and a mixing unit 490 that generates culture gas.
The sterilization box 471 includes a heater 471H that keeps the inside of the sterilization box 471 at a predetermined temperature, a culture solution bottle 472 that stores a culture solution therein, a reserve tank 473 that stores a reserve culture solution therein, and a used culture. Turns ON / OFF the main power of the waste liquid tank 474 for storing the liquid, the UV lamp 425 for sterilizing the inside of the sterilization box 471, the open / close door 475 used when the culture solution bottle 472 and the like are taken in and out of the sterilization box 471, and the culture unit 470. A main power switch 476 is provided.

The culture solution bottle 472 includes a culture solution circulation pipe 477 that circulates the culture solution to and from the incubator box 500, a supply pipe 478 that supplies a spare culture solution from the reserve tank 473, and a culture solution bottle 472 that has been used from the culture solution bottle 472. A waste liquid pipe 479 for discharging the culture liquid to the waste liquid tank 474 is disposed.
In addition, a culture solution pump 480 for sending the culture solution from the culture solution bottle 472 to the incubator box 500 and circulating the culture solution is disposed in the culture solution circulation pipe 477. Since the culture solution in the chamber 510 can be replaced with a new one by the culture solution pump 480, the period during which the cells CE can be cultured can be further extended as compared with the case where the culture solution cannot be replaced.
The supply pipe 481 is provided with a supply pump 481 for sending the culture solution from the reserve tank 473 to the culture solution bottle 472. The waste solution pipe 479 is a waste solution pump for sending the used culture solution from the culture solution bottle 472 to the waste solution tank 474. 482 is arranged.
As described above, the waste liquid tank 474 for storing the used culture medium may be used, or a discharge port for directly discharging the used culture liquid to the outside without using the waste liquid tank 474 may be provided. .

  The culture solution bottle 472 is provided with a culture solution temperature sensor (not shown) for detecting the temperature of the culture solution inside, and the output of the culture solution temperature sensor is input to the computer PC via the culture solution temperature detection unit 483. Are arranged as follows. The culture medium temperature data input to the computer PC is stored as text data in a memory and can be used for comparison / verification with the detection result of the cell CE.

In the heater 471H, a culture solution temperature control unit 484 for controlling the temperature of the culture solution via the temperature in the sterilization box 471 based on an instruction from the computer PC is disposed. The temperature of the culture solution supplied from the culture solution bottle 472 is maintained at approximately 37 ° C. by the culture solution temperature control unit 484, thereby preventing a decrease in the activity of the cells CE due to a change in the temperature of the culture solution. Further, on the wall surface of the culture unit 470, a temperature display unit 485 that displays the culture solution temperature detected by the culture solution temperature sensor is arranged.
The culture solution pump 480 is provided with a culture solution pump control unit 486 that controls the circulation of the culture solution based on instructions from the computer PC. The replenishment pump 481 and the waste liquid pump 482 are also arranged so that their operations are controlled based on instructions from the computer PC.

The UV lamp 425 is connected to a UV lamp switch 436 disposed on the wall surface of the culture unit 470. Between the UV lamp 425 and the UV lamp switch 436, a timer 437 for temporally controlling the operation of the UV lamp 425 is provided. Has been placed. Further, a sterilization indicator lamp (not shown) for displaying the lighting of the UV lamp 425 is arranged.
The UV lamp 425 is controlled separately from the main power switch 476, and can operate even when the main power is turned off.

  As shown in FIGS. 16 and 17, the mixing unit 490 includes a heater (not shown) that keeps the inside of the mixing unit 490 at a predetermined temperature, and the carbon dioxide gas concentration in the culture gas supplied to the incubator box 500. A culture gas mixing tank 491 to be adjusted and a CO2 pump 493 for supplying carbon dioxide gas from a CO2 tank 492 disposed outside the culture unit 470 are arranged in the culture gas mixing tank 491.

In the culture gas mixing tank 491, a CO2 concentration detector 494 for detecting the carbon dioxide gas concentration inside is arranged, and the output of the CO2 concentration detector 494 is arranged to be input to the computer PC. The CO2 pump 493 is provided with a CO2 concentration control unit 495 that controls the amount of carbon dioxide gas supplied to the culture gas mixing tank 491 based on an instruction from the computer PC. In addition, a CO2 concentration display unit 496 for displaying the carbon dioxide gas concentration in the culture gas mixing tank 491 detected by the CO2 concentration detection unit 494 is disposed on the wall surface of the culture unit 470.
Further, a culture gas supply pipe 497 is disposed between the culture gas mixing tank 491 and the culture solution bottle 472. Therefore, the culture gas is supplied to the culture solution via the culture gas supply pipe 497, and the culture gas can be sufficiently dissolved in the culture solution. Thus, by producing a culture solution in which a culture gas having a carbon dioxide gas concentration of 5% is dissolved in the culture solution bottle 472, the culture solution containing the culture gas and nutrients necessary for the growth of the cells CE are contained. Is supplied to a chamber 510 described later. In addition, the pH of the culture solution can be adjusted by dissolving the culture gas in the culture solution.
The carbon dioxide gas concentration input to the computer PC from the CO2 concentration detector 494 is stored as data in the memory and can be processed in the computer PC.

Next, an observation method in the biological sample observation system 410 having the above configuration will be described.
First, scanning method and detection range selection in the present embodiment will be described with reference to FIGS. 21 (a), (b), (c), and (d).
FIGS. 21A, 21B, 21C, and 21D are diagrams showing examples of scanning method and detection range selection in the present embodiment.

In the example shown in FIG. 21A, by specifying the upper left point a and the lower right point b of the measurement target range M on the displayed image, the measurement target range M (enclosed by a broken line in the figure). Range). Specifically, the measurement target range M may be set by dragging between the points a and b using a device such as a mouse, or the coordinate values of the points a and b are input and instructed. Also good.
The measurement part by the detector 449 is scanned in the left-right direction within the set measurement target range M as indicated by arrows in the figure. That is, when scanning from the left to the right in the figure, the scanning is performed in parallel with the X-axis direction, and when scanning from the right to the left, the scanning is performed obliquely downward to the left. Among these, imaging of the cell CE is performed when scanning from left to right.

FIG. 21B shows two examples of the measurement target range M set by the method described above. First, two measurement target ranges MA and MB are set by the method described above. The measurement target range MA and the measurement target range MB are set so that they are arranged at a predetermined interval in the X-axis direction in the drawing and all overlap in the Y-axis direction.
The measurement part by the detector 449 in this example is scanned so as to measure the measurement target ranges MA and MB in parallel, as indicated by arrows in the figure. That is, when scanning from left to right in the figure, the measurement target range MA is scanned from the measurement target range MB, and when scanning from right to left, the measurement target range MB is scanned from the measurement target range MA. Is done.

FIG. 21C is an example in which the measurement target ranges set by the above-described method are two examples, and the arrangement of the two measurement target ranges MA and MB is different. Here, the measurement target range MA and the measurement target range MB are set so as to be arranged at a predetermined interval in the X-axis direction in the figure and partially overlapped in the Y-axis direction in the figure.
In the measurement part by the detector 449 in this example, as indicated by the arrows in the figure, only the overlapping part in the Y-axis direction of the measurement target ranges MA and MB is continuously scanned. That is, first, a portion where the measurement target range MA does not overlap is scanned. Next, scanning of overlapping portions of the measurement target ranges MA and MB is continuously performed. Then, the non-overlapping portion of the measurement target range MB is scanned.

FIG. 21D shows two examples of the measurement target range M set by the above-described method, and the arrangement of the two measurement target ranges MA and MB is the same as that in FIG. Is a different example.
In the measurement part by the detector 449 in this example, the measurement target ranges MA and MB are separately scanned as indicated by arrows in the drawing. That is, first, the entire range of the measurement target range MA is scanned, and then the entire range of the measurement target range MB is scanned.

  Of the scanning methods shown in FIGS. 21A, 21B, 21C, and 21D described above, a scanning method that minimizes the total moving distance or scanning time is set by a set parameter described later and It is automatically selected by the computer PC based on the measurement mode.

In addition, when imaging the range in which cells are cultured, if the configuration allows the imaging to be divided into a plurality of areas (detection ranges) such as setting the measurement target range M in this way, imaging of only the necessary part is performed as necessary. Can be performed.
For example, if the setting of the computer PC is changed so that scanning of the entire range to be scanned and scanning of a predetermined part of the region are alternately performed, it is peculiar to biological samples that occur only for a short period of time. Can capture the phenomenon. As an example, when scanning of the entire range to be scanned is performed every 30 minutes, if scanning of a predetermined measurement target range M in which there are cells to be noticed is performed in between, only about 15 minutes are performed. It is also possible to capture that a unique phenomenon that does not occur has occurred in a cell of interest.
Further, since only the necessary measurement target range M is scanned when necessary, the scanning time can be shortened and the light irradiation time for other cells can be shortened.

Next, the procedure for measuring the cell CE will be described using each flowchart.
First, measurement parameters are set before the measurement of the cell CE. Therefore, the flow of setting the measurement parameters will be described with reference to FIG.
FIG. 22 is a flowchart for explaining the flow of setting measurement parameters.
First, measurement parameters are set (STEP 21).
Thereafter, default conditions are set (STEP 22). The conditions set here are, for example, culture conditions and measurement conditions such as a CO 2 concentration of 5% and a temperature of 37 ° C. These set conditions can be changed to predetermined conditions by the user.

Next, the measurement target is selected (STEP 23). The measurement target is a cell CE container such as a microplate 520 or a slide glass 516.
Next, the measurement mode is selected (STEP 24). Measurement modes include an area imaging mode, a line imaging mode, and an automatic mode. The automatic mode is a mode for automatically selecting a measurement mode with a short measurement time from other measurement modes.

Next, the measurement magnification is selected (STEP 25), and then the detection wavelength is selected (STEP 26). The measurement magnification and the detection wavelength can be selected from two or more options.
Here, as a method for selecting the detection wavelength, a list of fluorescent proteins to be used, for example, GFP, HC-Red, and the like is stored in advance in the computer PC and selected from the stored list. The computer PC automatically selects an excitation filter 456, an absorption filter 458, and the like that are optimal for observation based on the selected fluorescent protein. In this way, predetermined fluorescence can be detected from the cell CE.
Note that the excitation filter 456, absorption filter 458, objective lens 448, and the like during measurement are automatically changed in synchronization with driving of the X-axis operation stage 422X and Y-axis operation stage 422Y.

Next, a measurement interval is set (STEP 27).
Then, a preview screen is captured (STEP 28), and a preview image is displayed on the monitor (STEP 29). Here, when the user gives an instruction using a preview button or the like for instructing display of the preview image on the monitor, the preview image is displayed on the monitor. Then, the user can check the preview image displayed on the monitor.

Next, the measurement range is selected (STEP 30). After selecting the measurement range, the preview image may be displayed on the monitor again to check whether the measurement range is a predetermined range.
Next, a predetermined measurement interval is selected from the set plurality of measurement intervals (STEP 31).
When a measurement start switch (not shown) is pressed (STEP 32), the measurement of the cell CE is started (STEP 33). If the measurement start switch is not pressed, the process waits until the measurement start switch is pressed (STEP 32).

  In STEP 32, when the measurement start switch is not pressed, it may be set to be able to return to various predetermined STEPs so that various settings can be reset.

When the setting of the measurement parameters is completed, the cell CE is measured next. Accordingly, the flow of measurement of the cell CE will be described with reference to FIG.
FIG. 23 is a flowchart for explaining the flow of measurement.
First, when measurement is started, the measurement target range is read (STEP 41). Thereafter, the magnification is read (STEP 42), and the detection wavelength is read (STEP 43).
Next, the measurement mode is read (STEP 44). Here, the optimum stage scanning method is determined based on the read measurement object range, magnification, detection wavelength (fluorescence wavelength), and the like. When the measurement mode is set to the automatic mode, the imaging mode is also determined here.

Next, the operation methods such as the X-axis operation stage 422X and the Y-axis operation stage 422Y according to the determined stage scanning method are analyzed (STEP 45), and the analyzed operation method data (operation data) is stored in the computer PC. It is stored in a table (STEP 46).
Thereafter, measurement is performed by a different measurement method depending on whether or not the area sensor mode is selected (STEP 47).

First, the case where the area sensor mode is selected will be described.
When the measurement start switch is pressed, the X-axis operation stage 422X and the Y-axis operation stage 422Y are moved to the measurement start position (STEP 50). Here, the computer PC reads the input measurement start position, moves the X-axis operation stage 422X and the Y-axis operation stage 422Y to the measurement start position, and causes the cell CE to be positioned within the imaging field of the objective lens 448. Moved.
Then, the shutter 435 is set to OPEN (STEP 51), and the objective lens 448 is selected (STEP 52). Here, based on the measurement magnification set by the computer PC, the revolver 447 is driven to select the objective lens 448 having a predetermined magnification.

Next, the filter unit 445 is selected (STEP 53). Here, based on the fluorescent protein set by the computer PC, the filter control unit 446C selects an excitation filter 456, an absorption filter 458, and the like that are optimal for measurement.
The operations up to this point (from STEP 50 to STEP 53) after the above measurement start switch is pressed are automatically selected and executed in accordance with the measurement mode.
Thereafter, the focus position is detected (STEP 54), and the image is captured and output to the image memory unit of the computer PC (STEP 55).

If the acquisition of the necessary image is not completed, the operations from the selection of the objective lens 448 (STEP 52) to the image capture and the image data output to the image memory unit of the computer PC (STEP 55) are performed again. The process is repeated until the acquisition is completed (STEP 56).
When the necessary image acquisition is completed, the X-axis operation stage 422X or the Y-axis operation stage 422Y is driven one step (STEP 57). If the position to which the X-axis operation stage 422X or Y-axis operation stage 422Y has moved is within the measurement target range, the operation from the selection of the objective lens 448 (STEP 52) to the one-step stage drive (STEP 57) is repeated again. This repetitive operation is repeated until the position where the stage 422 has moved is outside the measurement target range (STEP 58).

When the movement destination of the X-axis operation stage 422X or the Y-axis operation stage 422Y is outside the measurement target range, the shutter 435 is turned off (STEP 59).
Thereafter, when a predetermined measurement time interval is reached, the operation from the time when the shutter 435 is again set to OPEN (STEP 51) to the time when the shutter 435 is closed (STEP 59) is repeated until the measurement time ends (STEP 60).

Next, a case where the area sensor mode is not selected will be described.
When the measurement start switch is pressed, the X-axis operation stage 422X and the Y-axis operation stage 422Y are moved to the measurement start position (STEP 70). Here, the computer PC reads the input measurement start position, moves the X-axis operation stage 422X and the Y-axis operation stage 422Y to the measurement start position, and causes the cell CE to be positioned within the imaging field of the objective lens 448. Moved.
Then, the shutter 435 is set to OPEN (STEP 71), and the focus position is detected (STEP 72).

Next, the objective lens 448 is selected (STEP 73). Here, based on the measurement magnification set by the computer PC, the revolver 447 is driven to select the objective lens 448 having a predetermined magnification.
Then, the filter unit 445 is selected (STEP 74). Here, based on the fluorescent protein set by the computer PC, the filter control unit 446C selects an excitation filter 456, an absorption filter 458, and the like that are optimal for measurement. The operations up to this point (from STEP 70 to STEP 74) after the measurement start switch is pressed are automatically selected and executed in accordance with the measurement mode.

Thereafter, driving of the X-axis operation stage 422X and the Y-axis operation stage 422Y is started (STEP 75), and image capture and image data output to the memory unit of the computer PC are performed (STEP 76).
If the necessary image acquisition has not been completed, the operations from the selection of the objective lens 448 (STEP 73) to the image capture and the image data output to the memory unit of the computer PC (STEP 76) are repeated. The process is repeated until the image acquisition is completed (STEP 77).

When the necessary image acquisition is completed, the shutter 435 is closed (STEP 78).
Thereafter, when a predetermined measurement time interval is reached, the operation from the time when the shutter 435 is again set to OPEN (STEP 71) to the time when the shutter 435 is closed (STEP 78) is repeated until the measurement time ends (STEP 79).

When the imaging of the cell CE is completed, the captured image is processed next. Therefore, a processing method of the captured image will be described with reference to FIG.
FIG. 24 is a flowchart illustrating an image processing method.
First, the image processing unit of the computer PC extracts a background image from the captured image stored in the memory unit (STEP 91) and removes the background image (background) from the captured image (STEP 92).
Next, the maximum luminance range of the image that can be enhanced is read (STEP 93), and the image is enhanced by multiplying a predetermined coefficient, for example, according to the maximum luminance range (STEP 94). By these processes, the image is enhanced so that each cell CE can be easily recognized in granular form from the image from which the background is removed.

Then, for example, by extracting a part having a luminance equal to or higher than a predetermined threshold from the emphasized image, each luminance of the cell CE is recognized as a clear granularity (STEP 95).
Next, geometric features such as the center of gravity and area of the cell CE, chemical features, and optical features such as fluorescence luminance are more accurately recognized and extracted in association with the location information of the cell CE. (STEP 96). By extracting these feature amounts, each cell CE can be identified.
After extracting the feature quantity of the cell CE, the enhancement work (STEP 94) performed for recognizing the cell CE is corrected (STEP 97). By this correction, the influence of the predetermined coefficient used for image enhancement is removed.
Next, the corrected feature amount is output to, for example, a file and stored in the file (STEP 98).

  Therefore, the image processing unit of the computer PC can image the fluorescence amount distribution of the cells CE at each position on the entire surface such as a slide glass or a microplate. In addition, since the image processing unit can accurately track each cell CE, for example, paying attention to only a predetermined number of cells CE, the fluorescence distribution inside the cells CE is locally long while culturing. It is also possible to measure time. Furthermore, while culturing the cell CE, for example, the entire surface of a slide glass, a microplate, or the like is measured at regular intervals, and the fluorescence amount of the cell CE with respect to time can be automatically measured.

Next, data processing performed after data such as the feature amount of the cell CE is extracted from the captured image will be described with reference to FIG.
FIG. 25 is a flowchart for explaining the flow of data processing.
Here, the data (feature value) of the cell CE accumulated in the file is processed by the data processing unit of the computer PC.
First, the data processing unit reads the raw data (feature amount) of the cells CE accumulated in the file (STEP 101), and rearranges the data so that the cells CE are arranged in time series (STEP 102). When the data is rearranged, the data processing unit graphs the luminance, that is, the change in the expression level with time for each cell CE (STEP 103).
When the graphing is completed, the data processing unit displays a preview of the graph (STEP 104), and outputs the graphed data to a file (STEP 105).

  By performing this treatment, it is possible to easily observe the temporal change of one cell when the cell CE is cultured for a long time. Accordingly, it is possible to accurately and easily measure a change in the expression level of the cell CE over time during the culture.

Next, adjustment of the irradiation light amount performed at the time of measuring the cell CE will be described with reference to FIG.
FIG. 26 is a flowchart for explaining the flow of light amount adjustment.
First, the irradiation light quantity irradiated to the cell CE is measured (STEP 111). The irradiation light amount may be calculated from the output of the light amount monitor 450, may be measured by providing an illuminometer, or may be calculated from the output of the power meter by providing a power meter.

If the measured irradiation light quantity is within the allowable range, the process returns to the measurement of the irradiation light quantity (STEP 111) again and is repeated until the irradiation light quantity falls outside the allowable range (STEP 112).
When the amount of irradiation light falls outside the allowable range, the ND filter (not shown) included in the light amount adjustment mechanism 443 is replaced (STEP 113), and the amount of irradiation light is adjusted to be within the allowable range. Thereafter, the process returns to the measurement of the irradiation light amount (STEP 111), and the adjustment of the irradiation light amount is repeated.

Next, a method for controlling the supply / exchange of the culture medium to the chamber 510 will be described with reference to FIG.
FIG. 27 is a flowchart for explaining a culture solution replenishment / exchange method.
First, the background value of the captured image is analyzed (STEP 121). Autofluorescence of the culture solution is imaged in the background of the captured image, and the brightness of the autofluorescence of the culture solution is analyzed.
Here, since the brightness of the autofluorescence increases as the culture solution becomes older, the replacement time of the culture solution can be detected by measuring the brightness of the autofluorescence.

If the temporal variation of the analyzed background value is within a predetermined specified value, the process returns to the background value analysis (STEP 121) again until the temporal variation of the background value becomes larger than the predetermined predetermined value. Repeated (STEP 122).
When the variation of the background value with time becomes larger than a predetermined specified value, the culture liquid waste pump 482 is driven (STEP 123), and then the culture liquid replenishment pump 481 is driven (STEP 124).

As described above, the time for replenishment / exchange of the culture solution may be determined by autofluorescence of the culture solution, may be performed continuously, or automatically at a time interval specified in advance by the user. Replenishment / exchange may be performed, or by selecting a cell CE from a pre-registered table, it may be possible to appropriately designate the replacement time of the culture solution. Also, the amount to be replaced may be set by the user, may be determined by the autofluorescence of the culture solution, or all of the culture solution in the chamber 510 may be replaced in a batch or registered in advance. By selecting the cell CE from the existing table, the exchange amount of the culture solution may be appropriately designated.
It may be set automatically by converting the weight.
In the present embodiment, the value of the background autofluorescence is detected using the captured image, but this detection may be detected from an image captured where a cell CE does not exist. For example, an optical detector may be provided in the vicinity of the culture solution bottle 472 for detection.
By the measurement procedure as described above, a cell tracking image representing a change in position of each cell over time as shown in FIG. 28 can be acquired.

Next, the procedure for culturing and measuring using the microplate 520 will be described with reference to FIG.
FIG. 29 is a flowchart illustrating a procedure for culturing and measuring using the microplate 520.
First, sterilized water is supplied to the water tank 521 of the incubator box 500a (STEP 131).
Next, after the computer PC is started (STEP 132), the main power of the detection unit 420 and the culture unit 470 is turned on (STEP 133).

  Thereafter, the internal fan 522 in the incubator box 500a is driven (STEP 134) to circulate the air in the incubator box 500a. Then, the CO2 concentration control unit 495 is activated (STEP 135) to control the carbon dioxide gas concentration of the culture gas supplied to the incubator box 500a to 5%. Thereafter, each temperature control unit is activated (STEP 136), and the temperature of the culture solution, the temperature of the culture gas, the temperature in the heat insulating box 421, and the like are controlled to approximately 37 ° C.

Thereafter, the open / close door 427 of the detection unit 420 is opened (STEP 137), the incubator box 500a is set on the stage 422 (STEP 138), and the open / close door 427 is closed (STEP 139).
Next, the transmission light source 423 is turned on (STEP 140), the cell CE is irradiated with the transmitted light, and the measurement conditions are set (STEP 141).

Then, the measurement of the cell CE is started by turning on the measurement start button (STEP 142).
First, a predetermined cell CE is scanned in advance to perform autofocus (STEP 143), and when the focal position of each part is determined, the shutter 435 is opened (STEP 144).
Next, an image of the cell CE is captured and output (STEP 145). Here, the captured image data is output to the memory unit of the computer PC.

  If the necessary image acquisition is not completed, the operations from autofocus (STEP 143) to image capture and output (STEP 145) are repeated until the necessary image acquisition is completed (STEP 146). Here, the necessary image is, for example, an image photographed at a selected wavelength or an image photographed at a selected magnification.

  When the necessary image acquisition is completed, the X-axis operation stage 422X or the Y-axis operation stage 422Y is driven one step (STEP 147). If the position to which the X-axis operation stage 422X or the Y-axis operation stage 422Y has moved is within the measurement target range, the operation from autofocus (STEP 143) to one-step stage drive (STEP 147) is repeated again. This repetitive operation is repeated until the position where the stage 422 has moved is outside the measurement target range (STEP 148).

When the movement destination of the X-axis operation stage 422X or the Y-axis operation stage 422Y is outside the measurement target range, the shutter 435 is closed (STEP 149), and the X-axis operation stage 422X and the Y-axis operation stage 422Y are moved to the home position ( (STEP 150).
Thereafter, when a predetermined measurement time interval is reached, the operation from the autofocus (STEP 143) to moving the stage to the home position again (STEP 150) is repeated until the measurement time ends (STEP 151).

When the measurement time ends (STEP 152), the door 427 is opened (STEP 153), and the microplate 520 is removed from the incubator box 500a (STEP 154). Then, sterilized water is removed from the water tank 521 (STEP 155), and the open / close door 427 is closed (STEP 156).
Thereafter, the UV lamp 425 in the heat insulation box 421 is turned on (STEP 157), sterilization in the heat insulation box 421 is performed, and the measurement is finished.

As described above, sterilization in the heat insulation box 421 may be put at the end of the measurement procedure, or may be put at the beginning of the measurement procedure and the heat insulation box 421 may be sterilized before the measurement.
Note that, as described above, autofocus may be performed for each measurement of the cell CE, or may not be performed for each measurement.

According to the above configuration, the temperature environment is maintained by the heat insulation box 421 and the humidity environment and the culture medium environment are maintained by the chamber 510 disposed in the heat insulation box 421. Under the influence of the temperature environment, the temperature environment is also maintained in the chamber 510.
Therefore, a rapid change or non-uniformity of the temperature environment that damages the cell CE is mitigated through the humidity environment and the culture medium environment, and therefore the damage to the cell CE is reduced. Can do.
Further, since the chamber 510 has a smaller volume than the heat retaining box 421, it is easy to maintain and control the humidity environment and the culture solution environment, and it is possible to make it difficult to damage the cells CE.

In addition, since the cell CE can be observed through the heat insulating box 421, the incubator box 500, and the chamber 510, it can be observed while culturing without damaging the cell CE. Therefore, the behavior in the cell CE occurring in the culture process can be measured accurately and with time.
For example, the response of the cell CE to be observed can be measured in real time while changing the culture conditions, and the presence or absence of protein expression, the measurement of the expression level, and the change in the expression level over time can be accurately measured. Can do.

  Furthermore, the activity of the cell CE can be prevented from being impaired by various operations in one observation, and the same cell CE can be observed a plurality of times. In addition, since the same cell CE can be observed multiple times at time intervals, there is no need to limit the experimental protocol.

In addition, since the detection unit 440 observes the cell CE in the chamber 510 via the heat insulation box 421 and the incubator box 500, it is not necessary to take the cell CE out of the chamber 510 during the observation. It can be fixed in the middle chamber 510. Therefore, the same position can be observed exactly every measurement. In addition, it is possible to prevent contamination during observation and to prevent the cell CE from being loaded.
Furthermore, it is possible to prevent the function of the detection unit 440 from being impaired by environmental conditions in the chamber 510 (for example, a combination of carbon dioxide gas and humidity).

  Further, since the cells CE are stored in the heat insulation box 421 and the chamber 510 disposed in the incubator box 500, the environment outside the cells CE and the incubator box 500 is compared with the case where the chamber 510 is not disposed. Can be secured. Therefore, the influence of an electric field, a magnetic field, and the like by the drive motor of the stage 422 outside the incubator box 500 received by the cell CE and the magnet provided on the open / close door 427 can be reduced.

[Fifth Embodiment]
Next, a fifth embodiment of the present invention will be described with reference to FIGS.
The basic configuration of the biological sample observation system of the present embodiment is the same as that of the fourth embodiment, but the configurations of the detection unit and the culture unit are different from those of the fourth embodiment. Therefore, in the present embodiment, only the periphery of the detection unit and the culture unit will be described using FIGS. 30 to 32, and description of the chamber and the like will be omitted.
FIG. 30A is a front view of the biological sample observation system in the present embodiment, and FIG. 30B is a side view of the biological sample observation system in the present embodiment.
As shown in FIG. 30, the biological sample observation system 600 is roughly composed of an inverted microscope (microscope imaging device) 610 and a culture stage 620. The inverted microscope 610 and the culture stage 620 may be fixed integrally or may be configured to be detachable.

If the culture stage 620 can be attached to and detached from the inverted microscope 610, an existing inverted microscope can be used. In this case, for example, due to restrictions on the shape and structure related to the attachment of the culture stage 620, even if a stage drive motor is disposed in the vicinity of the cell CE, the influence of quantity electricity and magnetism on the cell CE can be reduced.
Further, for example, when observing the cell CE using the inverted microscope 610, the culture stage 620 can be attached to the inverted microscope 610, and at other times (for example, when culturing the cell CE), it is inverted. The mold microscope 610 can be removed from the microscope.

FIG. 31 is a plan view of the culture stage 620, and FIG. 32 is a perspective view of the culture stage 620.
As shown in FIGS. 30 and 31, the culture stage 620 includes a housing 621, an opening / closing lid 622 provided on the upper surface of the housing 621, an X-axis operation stage 422X, a Y-axis operation stage 422Y, The belt heater 620 </ b> H, a heat radiating plate 623, a fan 624, and a culture gas supply connector 625 are roughly configured.

The housing 621 is preferably made of a light-shielding material with high anti-corrosion properties such as anodized aluminum or stainless steel such as SUS316, and more preferably a material with low thermal conductivity from the viewpoint of heat retention. It is good to select.
The inside of the housing 621 is divided into a measurement area 626 for observing cultured cells and a non-measurement area 627 for only culturing cells. In the culture stage 620, the cells are cultured, the microplate 520 holding the cells is housed inside, and the cells in the microplate 520 can be observed from the outside of the culture stage 620. . Here, as shown in FIG. 31 and FIG. 32, the case where a microplate 520 is used as a culture vessel will be described, but a dish or a flask can also be used.

A fan 624 and a culture gas supply connector 625 are arranged on the side wall in the non-measurement area 627 of the housing 621. Further, in a region where the fan 624 and the culture gas supply connector 625 are not disposed (including the measurement area 626), a heater 620H and a heat dissipation plate 623 that diffuses the heat of the heater 620H are disposed.
The fan 624 convects the air in the culture stage 620 and prevents direct air from hitting the incubator box 500a.
The temperature in the culture stage 620 is raised by the heater 620H and controlled to 36.5 ° C. ± 0.5 ° C.

As shown in FIGS. 31 and 32, an X-axis operation stage 422X and a Y-axis operation stage 422Y are installed on the bottom surface of the housing 621. The X-axis operation stage 422X and the Y-axis operation stage 422Y are driven by, for example, a motor and a ball screw.
A small or strip-shaped heater (not shown) is attached to the Y-axis operation stage 422Y. The heater is disposed at a position where the microplate 520 is evenly heated.

The measurement area 626 and the non-measurement area 627 are obtained by dividing the inside of the culture stage 620 in the X-axis direction by a top plate 628 fixed to the housing 621 and a pair of partition receivers 629. That is, in FIG. 31, the left area of the partition receiver 629 is a measurement area 626, and the right area is a non-measurement area 627.
In addition, a partition plate 629a formed in a shape that substantially matches the cross-sectional shape of the housing 621 is attached to the X-axis operation stage 422X.
The partition plate 629a moves the X-axis operation stage 422X to the non-measurement area 627 side, whereby the side surfaces of both end portions (end portions in the Y-axis direction) of the partition plate 629a come into contact with the partition receiver 629 and the housing 621. The inner space is divided into two in the left-right (X-axis) direction.
Furthermore, since the end of the partition plate 629a on the top plate 628 side is arranged so as to contact the lower surface of the top plate 628, the measurement area 626 and the non-measurement area 627 are made into two spaces divided from each other. Can do.

A glass upper lid 630 is detachably attached to the housing 621 in the upper opening area of the measurement area 626 and is disposed so as to cover the upper opening area of the measurement area 626. Examples of the method for attaching the glass upper lid 630 include a method for fixing the glass upper lid 630 to the housing 621 with a screw, and a method for attaching the glass upper lid 630 with a lock mechanism, a claw, a magnet, and the like.
The glass upper lid 630 may be formed of the glass plate 631 over the entire surface or substantially the entire surface excluding the surrounding frame portion, or may have a minimum area as long as the measurement is not hindered. For the glass plate 631, it is preferable to use an optical glass material coated on both sides with an antireflection film (AR coating) in order to suppress reflection of light during transmission observation and epi-illumination observation.

Note that the antireflection film may be coated on both surfaces of the glass plate 631 as described above, or may be coated on one surface of the glass plate 631.
The glass top cover 630 can be removed as necessary when performing various operations such as replacement of the objective lens of the inverted microscope 610 and internal cleaning of the measurement area 626.
Note that the glass top cover 630 may be provided with an observation hole into which the objective lens of the inverted microscope 610 is inserted. Further, a rubber sheet may be arranged in the observation hole to close the gap between the objective lens and the observation hole. The sheet is desirably arranged so as not to hinder the relative movement between the objective lens and the culture stage 620.

An opening / closing lid 622 is attached to the upper opening region of the non-measurement area 627 so as to be opened and closed using a hinge or the like. When the open / close lid 622 is closed, one end of the open / close lid 622 is in contact with and supported by the top plate 628.
The opening / closing lid 622 is entirely made of a light-shielding material (for example, the same material as the housing 621). Is provided.

The peephole is an opening (window) formed in the opening / closing lid 622, and the peephole cover 632 is formed of, for example, a glass plate or a resin plate having a low transmittance, and is fitted into the peephole. Further, the peephole cover 632 may be formed of the same light-shielding material as the opening / closing lid 622, and may be attached detachably or closably.
The UV irradiation hole is an opening (window) formed in the opening / closing lid 622, and the UV irradiation hole cover 633 is formed of, for example, the same light-shielding member as the opening / closing lid 622, and is attachable / detachable to / from the UV irradiation hole. It is attached so that it can be opened and closed.
The UV irradiation hole cover 633 is removed when the non-measurement area 627 is irradiated with ultraviolet rays to be sterilized. A handy-type UV lamp can be used for UV light irradiation.

An incubator box 500a containing a microplate 520 is held on the Y-axis operation stage 422Y. Since the incubator box 500a is the same as that described in the fourth embodiment, the same components are denoted by the same reference numerals, and the description thereof is omitted.
The culture gas is supplied to the connector 523 of the incubator box 500a from the culture gas supply connector 625 of the culture stage 620 via the culture gas supply tube 634.

According to the above configuration, the biological sample observation system 600 according to the present invention is provided with the inverted microscope 610 so that the biological sample can be observed using the inverted microscope 610. Therefore, finer observation can be performed as compared with the case where the inverted microscope 610 is not provided.
It should be noted that the whole biological sample observation system 600 may be covered with a black screen or the like in order to prevent the cells from being affected by ambient light during cell measurement and culture.
In addition to cells, various biological samples such as bacteria, microorganisms, and eggs can be used as biological samples for measurement.

It is a figure explaining the imaging operation of a TDI system. It is a figure explaining transfer of the signal charge of a TDI system. It is a figure which shows the intensity | strength of the signal charge accumulate | stored in the horizontal line. It is a figure which shows the whole structure of the microscope imaging device in 1st Embodiment. It is a figure explaining the structure of a sample. It is a figure explaining the scanning in a TDI system. It is a flowchart which shows the measurement procedure in 1st Embodiment. It is a figure which shows the correlation of the brightness | luminance of a sample, and an output level. It is a time chart of the electronic shutter in a TDI system. It is a figure explaining another structural example of a sample. It is a figure which shows the whole structure of the microscope imaging device in 2nd Embodiment. It is a flowchart which shows the measurement procedure in 2nd Embodiment. It is a correlation diagram between the number of saturated pixels and the exposure time in the next imaging. It is a figure which shows the whole structure of the microscope imaging device in 3rd Embodiment. It is a figure explaining the captured image in 3rd Embodiment. It is a perspective view which shows the biological sample observation system provided with the microscope imaging device in 4th Embodiment. It is the schematic which shows the system configuration | structure of the biological sample observation system shown in FIG. It is a perspective view which shows the incubator box of the biological sample observation system shown in FIG. It is sectional drawing of the chamber of the incubator box shown in FIG. It is a perspective view which shows another example of the incubator box shown in FIG. It is a figure which shows the example of selection of the scanning method and detection range in a biological sample observation system. It is a flowchart which shows the flow of the setting of the measurement parameter in a biological sample observation system. It is a flowchart which shows the flow of a measurement in a biological sample observation system. It is a flowchart which shows the image processing method in a biological sample observation system. It is a flowchart which shows the flow of the data processing in a biological sample observation system. It is a flowchart which shows the flow of adjustment of the light quantity in a biological sample observation system. It is a flowchart which shows the replenishment / exchange method of the culture solution in a biological sample observation system. It is a figure which shows the cell tracking image showing the time-dependent change of the cell. It is a flowchart which shows culture | cultivation and a measurement using a microplate. It is the front view and side view which show the biological sample observation system provided with the microscope imaging device in 5th Embodiment. It is a top view of the culture | cultivation stage of the biological sample observation system shown in FIG. It is a perspective view of the culture | cultivation stage of the biological sample observation system shown in FIG.

Explanation of symbols

10, 110, 210 Microscope imaging device 20 Sample (inspection object)
22 Specimen Placement Unit 30, 422 Stage 31 Stage Drive Mechanism (Moving means)
40 Illumination device (illumination means)
49, 448 Objective lens 60, 160, 260 Imaging device (imaging means)
63, 63a, 263 Image sensor 167 Exposure time input section (exposure time input means)
410, 600 Biological sample observation system 440 Detection unit (microscope imaging device)
422X X-axis operation stage (stage)
422Y Y-axis operation stage (stage)
423 Transmitted light source (illumination means)
429 stage scanning unit (moving means)
441X, 441Y Epi-illumination light source (illumination means)
449 Detector (imaging means)
510 Chamber (inspection object)
520 Microplate (inspection object)
610 Inverted microscope (microscope imaging device)
S1, S3, S11, S13 Scanning (imaging)
S4, S14 Combined into one image

Claims (5)

A stage for holding the inspection object, an illuminating means for illuminating the inspection object, an imaging means for capturing an image of the inspection object, a moving means for relatively moving the stage and the imaging means, With
The imaging means generates accumulated charges corresponding to the object to be inspected, transfers the accumulated charges corresponding to the relative movement between the stage and the imaging means, and sequentially adds the accumulated charges to accumulate.・ Equipped with an image sensor that can capture images using the delay integration method.
When the imaging means captures the inspection object a plurality of times to obtain a plurality of images,
Each time a different image is acquired, the exposure time for generating the accumulated charge in the image sensor is changed,
A microscope imaging apparatus for synthesizing the plurality of images into one image. Exposure time input means for inputting the exposure time from the outside,
The imaging means performs imaging of the inspection object using the input exposure time,
The microscope imaging apparatus according to claim 1, wherein the length of the exposure time used for the next imaging is determined based on an image acquired by imaging using the input exposure time.   The microscope imaging apparatus according to claim 2, wherein the exposure time input by the exposure time input unit is a longest exposure time that is an upper limit of the exposure time.   The microscope imaging apparatus according to any one of claims 1 to 3, wherein the imaging unit includes an imaging element having a smear suppressing function.   A biological sample observation system comprising the microscope imaging apparatus according to claim 1.

Images