JP2015129713A - image acquisition device and method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide image acquisition device and a method for acquiring an image of a biological sample.SOLUTION: An image acquisition device includes a darkroom and a light source 34 that is provided inside the darkroom and irradiates a biological sample 21 held inside the darkroom with infrared light. The image acquisition device further includes an image acquisition unit that acquires a bright field image 41 of the biological sample 21 in an illuminated state where the biological sample 21 is irradiated with the infrared light and acquires a weak light image of the biological sample 21 in a non-illuminated state, and a control device 12 that has a display unit 13 displaying the bright field image 41 and the weak light image 42 overlapped with each other.

Description

  The present invention relates to an image acquisition apparatus and method for acquiring an image of a biological sample.

  In a method of analyzing a sample derived from a living organism such as a cell (biological sample) under a microscope, there is a method of performing analysis by superimposing a bright-field image and a luminescent image (dark-field image). In this analysis method, a bright field image is acquired by focusing the sample in a state in which the sample is irradiated with visible light. Similarly, photographing is performed over a predetermined exposure time in a dark field state where no visible light is applied to the sample, and a light emission image in which self-emission light or the like of the sample is captured is acquired. Then, the bright-field image and the light-emitting image acquired in the two steps are superimposed and used for, for example, applications such as cell identification or cell pickup work (see, for example, Patent Documents 1 and 2).

JP 2007-93488 A JP 2011-196867 A

  For example, when analyzing a so-called photosensitive biological sample such as a cell expressing an opsin gene, if the biological sample is irradiated with visible light to obtain a bright field image, the properties of the biological sample change. For this reason, the technique of irradiating visible light and acquiring a bright field image cannot be applied to the analysis of a photosensitive biological sample.

  On the other hand, it is difficult to focus on a biological sample under a dark field. The self-emission of a biological sample is usually extremely weak, and an exposure time of several minutes to several tens of minutes is required to acquire a luminescent image. If the focus is poor after the emission image is acquired, the emission position is acquired again after finely adjusting the focus position. Such an operation is repeated a plurality of times to adjust the focus. As a result, the focus adjustment under the dark field takes a lot of time, or the focus adjustment is not successful and there is a problem that a clear image cannot be obtained.

  The objective of this invention is providing the image acquisition apparatus which improved the convenience at the time of handling the photosensitivity biological sample.

  In order to achieve the above object, a microscope apparatus according to one aspect of the present invention includes a dark room, and a light source that is provided in the dark room and that irradiates a biological sample held in the dark room with infrared light. . The microscope apparatus acquires a bright field image from the biological sample in an illumination state where the infrared light is irradiated on the biological sample, and an image acquisition unit that acquires a weak light image of the biological sample in a non-illuminated state; A control unit having a display unit that displays the bright field image and the weak light image in a superimposed manner.

  According to said structure, the image acquisition apparatus which improved the convenience at the time of handling a photosensitive biological sample can be provided.

The perspective view which showed the whole structure of the microscope system concerning 1st Embodiment. The block diagram which shows the microscope apparatus of the microscope system shown in FIG. The graph which shows Ca ^<2+> reaction of the photosensitivity biological sample at the time of irradiating visible light and stimulation light using the conventional microscope apparatus which acquires a bright field image using visible light. The graph which shows Ca ^<2+> reaction of the photosensitivity biological sample at the time of irradiating infrared light and stimulation light using the microscope system of embodiment. The figure which shows the bright field image of the biological sample acquired using the infrared light in the microscope system shown in FIG. The figure which shows the weak light image (luminescence image) of the biological sample acquired in the dark field in the microscope system shown in FIG. The figure which shows the superimposition image displayed on the display part by superimposing the bright field image shown in FIG. 5, and the weak light image shown in FIG. The figure which shows the image acquired by irradiation intensity 1 using the microscope system of 2nd Embodiment. The figure which shows the image acquired by irradiation intensity 2 using the microscope system of 2nd Embodiment. The figure which shows the image acquired by irradiation intensity 5 using the microscope system of 2nd Embodiment. The figure which shows the image acquired by irradiation intensity 7 using the microscope system of 2nd Embodiment. The figure which shows the image acquired with the irradiation intensity | strength 10 using the microscope system of 2nd Embodiment. The graph which showed the brightness | luminance of the luminescent cell, the nonluminous cell, and background in the image acquired with the irradiation intensity 0 thru | or 10 using the microscope system of 2nd Embodiment, and a bright field image. The graph which shows the ratio of the brightness | luminance of the self-luminescence of a luminescent cell with respect to the brightness | luminance of a background, and the ratio of the brightness | luminance of a nonluminous cell from the data shown in the graph shown in FIG.

[First Embodiment]
Hereinafter, the microscope system according to the first embodiment will be described with reference to FIGS. 1 to 7. For example, the microscope system 11 cultivates a biological sample (cultured cells) using a container such as a dish, or acquires a weak light image and a bright field image (including a phase difference image) of the cells over time. Or an image (live image) showing the current state of the cell, or a single cell, a colony of cells, or an aggregate of cells based on the weak light image, bright field image, live image, etc. The embryoid body is picked up and used for multiple purposes. In the microscope system 11, the bright field image and the weak light image can be superimposed and displayed on the display unit 13 (display screen 14) of the control device 12. The microscope system 11 is an example of an image acquisition device.

  In the biological samples referred to in the following embodiments, various cells (embryonic stem cells (ES cells), tissue stem cells, induced pluripotent stem cells (iPS) into which luciferase, other luminescent or fluorescent proteins, etc. are introduced. Cells) and other living organisms into which luciferase or other luminescent or fluorescent proteins have been introduced. The weak light image referred to in the present embodiment includes a luminescent image obtained by self-luminous properties such as intracellular proteins (for example, various luciferases). The weak light image referred to in the present embodiment may include, for example, a fluorescent image obtained by irradiating a cell into which various proteins such as GFP are introduced with excitation light.

  The microscope system 11 includes a microscope device 15 and a peripheral device 16 attached to the microscope device 15. The microscope apparatus 15 includes, for example, an image sensor (CCD, CMOS, etc.) that exposes light for a sufficient time (1 minute or more) and a high NA imaging optical system (objective lens, imaging lens, etc.). A weak light image is acquired based on the weak light emitted from a cell or the like.

  As shown in FIGS. 1 and 2, the microscope apparatus 15 includes a main body 22 that can hold a biological sample 21 placed on a dish, a dark box 23 (dark room) provided on the top of the main body 22, and a dark box 23. A lid 24 that covers the upper portion of the plate, an incubator 25 provided in the dark box 23, a stage 26 in the incubator 25 having a dish shape, a container 27 such as a dish placed on the stage 26, and a container 27 And a biological sample 21 held in the container.

  The incubator 25 and the stage 26 are provided with observation holes. As shown in FIG. 1, the microscope apparatus 15 has a handle 28 for manually moving the stage in the X-axis direction (front-rear direction) and the Y-axis direction (left-right direction), for example, below the dark box 23. ing.

  The microscope apparatus 15 includes an objective optical system 31 provided below the dark box 23 of the main body 22, a first filter 32 that blocks unnecessary light passing through the objective optical system 31, and an objective optical system. 31 and a CCD camera 33 (color CCD camera) provided below the first filter 32 and capable of detecting light passing through the objective optical system 31. The objective optical system 31 may be provided with a zoom mechanism (not shown). The CCD camera 33 is an example of an image acquisition unit that can acquire images (a weak light image and a bright field image of the biological sample 21, a live image of the biological sample 21, etc.). As the CCD camera 33, a color CCD camera from which a so-called IR cut filter is removed is used.

  The peripheral device 16 connected to the microscope device 15 includes a light source 34 used when acquiring a bright field image and a fluorescent image, and a control device 12 (computer) connected to the light source 34 and the CCD camera 33. included.

  The light source 34 includes a lamp or LED that can irradiate the biological sample 21 held in the dark box 23 with infrared light. As the light source 34, it is preferable to use an infrared light emitting diode capable of irradiating near infrared light among infrared light. Infrared light generally refers to electromagnetic waves in the range from 0.78 μm to 1000 μm. Near-infrared light generally refers to electromagnetic waves in the range of 0.78 μm to 2 μm. In the present embodiment, an infrared light emitting diode having a maximum emission wavelength of 0.90 μm to 1.00 μm is used as an example of the light source 34.

  The light source 34 may have a lamp, LED, etc. which can irradiate white light separately. In the present embodiment, the light source 34 is controlled to be turned on / off under the control of the control device 12, but a switch may be provided to manually turn on / off.

  For example, the light source 34 is provided in the dark box 23 (in the lid 24), but the light source 34 is not necessarily mounted in the microscope apparatus 15. The light source 34 may be installed outside the microscope apparatus 15 and may be incident on the dark box 23 of the microscope apparatus 15 so that light from the light source 34 is transmitted by a mirror, an optical fiber, or the like. The light source 34 may further include a fluorescent unit capable of irradiating excitation light used during fluorescence observation. The microscope apparatus 15 that can be used in the present embodiment includes, for example, a light-emitting imaging system LUMINOWVIEW LV200 (LUMINOWVIEW (registered trademark) manufactured by Olympus Corporation, but is not limited to this, and other microscope apparatuses are used. May be.

  The control device 12 shown in FIG. 1 is a computer such as a general personal computer, and includes a display unit 13 (display screen 14) constituted by a liquid crystal display and the like, a keyboard 35 and a mouse 36 as input means, and the like. Contains. The control device 12 can control the photographing conditions of the CCD camera 33, convert the acquired image into an image and display it on the display unit 13, and control the amount of light from the light source 34. In addition to storing the acquired weak light image and bright field image, the control device 12 can perform image processing and image analysis of the weak light image and bright field image. The control device 12 can store data of the results of the image analysis together with the observation conditions. The control device 12 can perform control to switch the first filter 32 to an appropriate filter of a different type. Furthermore, the control device 12 can perform zoom control of the objective optical system 31.

  When acquiring a bright-field image with the microscope device 15, the biological sample 21 is illuminated so that near-infrared light is emitted from the light source 34, and the transmitted light travels through the objective optical system 31, and the first The light is not blocked by the filter 32 and is detected by the CCD camera 33. The light detected by the CCD camera 33 is sent to the control device 12 and imaged. When acquiring a weak light image, the light emitted from the biological sample 21 proceeds to the objective optical system 31, and unnecessary light is blocked by the first filter 32 and detected by the CCD camera 33. The light detected by the CCD camera 33 is sent to the control device 12 and imaged. When acquiring a fluorescent image, the light emitted from the light source 34 is transmitted through only light having a wavelength that excites the fluorescent protein or the like by a filter provided in the light source 34 and is irradiated on the biological sample 21. The light emitted from the biological sample 21 travels through the objective optical system 31, unnecessary light is blocked by the first filter 32, and is detected by the CCD camera 33. The light detected by the CCD camera 33 is sent to the control device 12 and imaged.

  Note that the bright-field image acquired using infrared light (particularly near-infrared light) as in this embodiment is a bright-field image acquired using visible light, although the background color is light purple. It has the same or better image quality. Rather, the bright-field image acquired using near-infrared light obtains a clearer image than the bright-field image acquired with visible light due to the high transmittance of near-infrared light when the biological sample 21 is thick. be able to.

  Subsequently, referring to FIG. 3 and FIG. 4, a case where observation is performed using a conventional microscope apparatus that acquires a bright-field image using visible light, and bright light using infrared light as in the present embodiment. A description will be given of the results of evaluating the influence of the both on the light-sensitive biological sample 21 when observed using the microscope apparatus 15 that acquires the field image.

Examples of the light-sensitive biological sample 21 include various cells (cultured cells) and biological tissues, or animals having eyes and sensory organs that can sense light. In this evaluation, for example, an opsin gene is expressed. Cultured cells (HEK293 cells) were used. In this cultured cell, OPN5 and Ca ²⁺ luminescence indicator expression vector (cpGL-CaM) have been introduced in advance by a predetermined method. This cultured cell is previously cultured in a medium supplemented with a retinal derivative. Prior to observation of luminescence of cultured cells, luciferin is added to the cultured cells at an optimal concentration.

In this experimental system for observing a photosensitive biological sample, first, the biological sample 21 is observed with light for cell observation (visible light or infrared light) for 20 seconds, and then OPN5 stimulation light (blue light) is applied to the biological sample 21. Was irradiated for 20 seconds and the Ca ²⁺ response was observed. In this experimental system, when stimulating light (blue light) is irradiated to a photosensitive cultured cell, Ca ²⁺ is released and the amount of luminescence of the cell decreases.

As shown in FIG. 3, when visible light is used as the cell observation light, the luminescence amount (relative luminescence amount) of the cultured cells decreases both during irradiation with visible light and irradiation with stimulation light. ^{A 2+} response was seen. On the other hand, as shown in FIG. 4, when infrared light is used as cell observation light, the light emission amount does not decrease when irradiated with infrared light, and the light emission amount decreases when irradiated with stimulation light. (Ie, a Ca ²⁺ response was seen). From this evaluation result, it was confirmed that the biological sample 21 can be evaluated without giving a light stimulus to the biological sample 21 even in an experimental system for evaluating the photosensitive biological sample 21.

  Subsequently, an observation procedure of the biological sample 21 using the microscope apparatus 15 of the present embodiment and a method for displaying a superimposed image will be described.

  After the biological sample 21 is installed in the microscope apparatus 15, the observation conditions are set according to the type of the biological sample 21 and the culture conditions. Subsequently, in a state in which the biological sample 21 is irradiated with infrared light, the CCD camera 33 is operated from the control device 12 to focus on the biological sample 21 to obtain a bright field image 41 (see FIG. 5). The bright field image 41 is stored in a storage device built in the control device 12.

  Subsequently, the weak light image 42 of the biological sample 21 is acquired in the focused state when the bright field image 41 is acquired. When acquiring a luminescent image as the weak light image 42, since the amount of luminescence from the biological sample 21 is extremely small, a clear image is acquired (captured) with sufficient exposure time. The exposure time is, for example, several minutes to several tens of minutes. When a fluorescent image is acquired as the weak light image 42, a sufficient amount of light emission can be obtained by irradiating the biological sample 21 with an optimal amount of excitation light, so that a clear image can be acquired in a relatively short time.

  The weak light image 42 is obtained by operating the CCD camera 33 from the control device 12 (see FIG. 6). The acquired luminescent image or fluorescent image is stored in a storage device built in the control device 12.

  Subsequently, a superimposed image obtained by superimposing the bright field image 41 and the weak light image 42 is displayed on the display unit 13. First, the user causes the display unit 13 to display the bright field image 41 that is desired to be superimposed and displayed. The bright field image may be selected from those stored in the storage device in the above process, or the biological sample 21 is again irradiated with infrared light (near infrared light) and the bright field image 41 or the weak field image is weak. It is also possible to acquire a live image at the same focus position as when the optical image 42 is acquired and display the live image on the display unit 13 as a bright field image.

  In a state where the bright field image 41 is displayed, the display is set to display an overlapping display, and the user is the same as a part of the biological sample 21 (colony, cell, part of animal, etc.) included in the bright field image 41. When the control device 12 is instructed to display the weak light image 42 including the portion, the control device 12 reads the weak light image 42. As a result, the display unit 13 displays the weak light image 42 so as to be superimposed on a part of the bright field image 41, and the display unit 13 can change the display position manually via the mouse 36 or the like. Is displayed. That is, in the present embodiment, the user uses the mouse 36 or the like so that the partial image of the biological sample 21 in the weak light image 42 overlaps the partial image of the corresponding biological sample 21 in the bright field image 41. Then, the faint light image 42 is moved manually, and the faint light image 42 is arranged with fine adjustment. The superposition of the weak light image 42 on the bright field image 41 may be automatically performed by the control device 12 by a method such as image recognition. Note that when the magnification of the bright field image 41 is changed, the control device 12 can appropriately change the reduction rate of the weak light image 42 in accordance with the magnification and display the superimposed image on the bright field image 41.

  As shown in FIG. 7, when the weak light image 42 is displayed at an appropriate position with respect to the bright field image 41, the position of the weak light image 42 is fixed with respect to the bright field image 41. 43 is created, and the superimposed image 43 is stored in the storage device of the control device 12 as necessary. The superimposed image 43 can be used for various purposes such as selection or pickup of cells expressing a specific gene, identification of differentiated cells, and the like.

  According to the first embodiment, the image acquisition device includes a dark room, a light source 34 that is provided in the dark room and is irradiated with infrared light on the biological sample 21 that is held in the dark room, and the red light is applied to the biological sample 21. An image acquisition unit that acquires the bright field image 41 from the biological sample 21 in the illumination state irradiated with external light, and acquires the weak light image 42 of the biological sample 21 in the non-illumination state, and the bright field image 41 and the weak light image. And the control device 12 having the display unit 13 that displays the image 42 in a superimposed manner.

  The image acquisition method of this embodiment irradiates the biological sample 21 with infrared light to acquire the bright field image 41 of the biological sample 21, acquires the weak light image 42 of the biological sample 21, and weakly matches the bright field image 41. The optical image 42 is superimposed and displayed.

  In general, it is difficult to focus on a biological sample such as a cell in a dark field, and the bright field image 41 is usually acquired before the weak light image 42 is acquired. In particular, when acquiring a luminescent image obtained by the self-luminous property of the cell as the weak light image 42, it is more difficult to focus because long-time exposure is required to acquire the luminescent image. .

According to said structure, the bright field image 41 of the biological sample 21 can be acquired, without giving a light stimulus with respect to what is called a photosensitive biological sample. Thus, for example, in an experimental system in which the biological sample 21 reacts to light stimulation, particularly in an experimental system in which the biological sample 21 changes irreversibly due to light stimulation, the bright field image 41 is acquired with infrared light. Rapid focusing and observation area determination can be performed. In addition, by irradiating the biological sample 21 with infrared light (near infrared light), due to high transparency of infrared light (near infrared light) (property that the surrounding material does not absorb near infrared light), A bright field image 41 with a clear outline of the biological sample 21 (cells) can be obtained. Thereby, a clear bright-field image can be obtained particularly in the biological sample 21 having a large thickness.
[Second Embodiment]
Subsequently, a second embodiment of the microscope system 11 will be described with reference to FIGS. The microscope system 11 of the second embodiment is different from that of the first embodiment in that the bright field image 41 including the self-luminous light of the biological sample 21 is acquired at one time. Common to the first embodiment. For this reason, mainly different parts will be described, and description of common parts will be omitted. The microscope system 11 of the second embodiment has the same appearance as that shown in FIG. The microscope system 11 is an example of an image acquisition device.

  Peripheral devices of the microscope system 11 include a light source 34 used when acquiring the bright field image 41 and a control device 12 (computer) connected to the light source 34 and the CCD camera 33 (image acquisition unit). It is.

  The light source 34 has a lamp or LED that can irradiate the biological sample 21 with infrared light. In particular, the light source 34 is preferably an infrared light emitting diode capable of irradiating near infrared light.

  The light source 34 is turned on for a short time during a part of the total exposure time when the bright field image 41 is acquired, and as a result, weak infrared light (near infrared light) is applied to the biological sample 21. Can be irradiated. At this time, weak lighting is realized by setting the lighting time of the light source to 0.5 to 1 second, for example, and lighting the light source a plurality of times during the exposure time. This weak infrared light is, for example, on the display screen 14 of the display unit 13 (in the bright field image 41 including the self-luminous light of the biological sample 21), and the background luminance is the self-luminous of the biological sample 21. The light is so weak that it is 1.25 times darker than the brightness.

  The on / off control of the light source is performed by, for example, the control device 12 connected to the light source 34, but the on / off switching operation may be manually performed by a switch connected to the light source 34. Further, weak infrared light may be realized by continuously lighting extremely weak infrared light (near infrared light) over the entire exposure time.

  Next, a procedure for acquiring the bright field image 41 including the self-luminous light of the biological sample 21 using the microscope system 11 of the present embodiment will be described.

  Prior to the acquisition of the bright field image 41 including the self-luminous light of the biological sample 21, it is desired to observe the biological sample 21 by operating the objective optical system 31 and the handle 28 in a state where the biological sample 21 is irradiated with infrared light. Focus on the point.

  Subsequently, a bright field image 41 including self-luminous light of the biological sample 21 is acquired by the CCD camera 33. Usually, since the self-luminous light of the biological sample 21 is weak, the acquisition of such a bright field image 41 requires several minutes to several tens of minutes of exposure time. In the present embodiment, the exposure time is, for example, 3 minutes. During the exposure time, the control device 12 performs on / off control of the light source 34 to irradiate the biological sample 21 with weak infrared light. In the present embodiment, the number of times the light source 34 is turned on during the exposure time is, for example, two. By such a procedure, for example, a bright field image 41 including self-luminous light of the biological sample 21 as shown in FIG. 9 can be acquired.

  Furthermore, the inventors examined how much weak infrared light intensity should be set. In FIG. 8 to FIG. 14, the optimum intensity of light is detected by manipulating the irradiation intensity of weak infrared light by changing the number of times the light source 34 is turned on during the exposure time. In this examination, the irradiation intensity is 1 when the light source 34 is turned on once, and the irradiation intensity n when the light source 34 is turned on n times. When the number of lighting times of the light source 34 is zero, the irradiation intensity is zero. In this way, the bright field image 41 including the self-luminous light of the biological sample 21 was obtained with the CCD camera 33 under the conditions of the irradiation intensity of 0 to 10.

  FIGS. 8 to 12 show images acquired at irradiation intensities of 1, 2, 5, 7, and 10, respectively. In all these images, the circled regions 1 to 3 represent cells that are self-luminous. Regions 4 to 6 represent non-luminescent cells, and regions 7 to 9 represent background. FIG. 13 is a table showing the self-luminous luminance, the non-luminous cell luminance, and the background luminance of the biological sample 21 (luminous cells) in each image of irradiation intensity 1 to 10 displayed on the display screen 14. Show. FIG. 14 shows the ratio of the self-luminous luminance of the biological sample 21 (luminescent cell) to the background luminance and the ratio of the non-luminous cell luminance to the background luminance.

  As a result of examining each image with irradiation intensity 1 to 10, self-luminescence (luminescent cells) of the biological sample 21 could be confirmed in the bright field image 41 for the images with irradiation intensity 1 and 2. On the other hand, as shown in FIG. 10 to FIG. 12, for images with an irradiation intensity of 3 to 10, the background brightness is increased, making it difficult to distinguish between luminescent cells and non-luminescent cells.

  As shown in FIG. 14, non-luminescent cells have a brightness of about 1.25 times the background brightness. And in the image of the irradiation intensity | strengths 1 and 2 which can distinguish a luminescent cell clearly as mentioned above, the brightness | luminance of the self-light emission of the biological sample 21 with respect to the brightness | luminance of a background showed the numerical value higher than 1.25 times in all. .

  For this reason, in order to acquire the bright field image 41 including the self-luminous light of the biological sample 21, the background luminance is 1.25 times or more darker than the luminance of the self-luminous (luminescent cell) of the biological sample 21. It was found that it is preferable to irradiate weak infrared light. Moreover, it can be said that the lower limit of the intensity of the infrared light is an intensity at which the contour of the biological sample 21 (luminescent cell) can be grasped.

  According to this embodiment, the image acquisition apparatus includes a dark room, a light source 34 that is provided in the dark room and that irradiates the biological sample 21 held in the dark room with weak infrared light, and the weak infrared light. An image acquisition unit that acquires a bright field image 41 including self-luminous light of the biological sample 21 from the biological sample 21 irradiated with the light.

  Further, in the method of the present embodiment, the bright light image 41 including the self-luminous light of the biological sample 21 is acquired by irradiating the biological sample 21 with weak infrared light.

  According to these structures, the process for superimposing the bright-field image 41 and the weak light image 42 as in the first embodiment is not required, and the procedure for obtaining the superimposed image can be simplified. Similarly, in the first embodiment, there is a time lag between when the bright field image 41 is acquired and when the weak light image 42 is acquired, and in the case of the moving biological sample 21 (for example, an animal). May cause a shift between the two images. According to this embodiment, even if the biological sample 21 is moving, the bright field image 41 (superimposed image) including the self-luminous light of the biological sample 21 can be easily acquired without deviation.

  The weak infrared light is so weak that the background luminance is 1.25 times or more darker than the self-luminous luminance of the biological sample 21. According to this configuration, it is possible to acquire the bright field image 41 in which the outline of the biological sample 21 is clarified by infrared light within a range where the self-emission of the biological sample 21 is not buried in the background light.

  The light source 34 realizes the weak infrared light by irradiating the biological sample 21 with infrared light during a part of the exposure time when acquiring the weak light image 42. According to this configuration, weak infrared light can be easily realized at low cost. The infrared light is near infrared light. For this reason, a clear image of the biological sample 21 can be obtained due to the high transmittance of near infrared light. For this reason, even when the biological sample 21 has some thickness, a clear image that transparently shows the outline of the biological sample 21 can be obtained.

  The microscope system 11 described in the present embodiment can be implemented with various modifications without departing from the spirit of the invention. For example, the microscope device 15 is provided with an incubator 25 for keeping cells, tissues, and the like as the biological sample 21 alive. However, the microscope device 15 may be applied to the microscope device 15 without such an incubator 25. it can. In the above-described embodiment, the bright field image 41 is described as being distinguished from the live image. However, the bright field image 41 may be replaced with a live image. Furthermore, one invention can be configured by combining the components in the first embodiment and the components in the second embodiment.

DESCRIPTION OF SYMBOLS 11 ... Microscope system, 12 ... Control apparatus, 13 ... Display part, 14 ... Display screen, 21 ... Biological sample, 23 ... Dark box, 33 ... CCD camera, 34 ... Light source, 41 ... Bright field image, 42 ... Low light image, 43 ... superimposed image

Claims (10)

A dark room,
A light source for irradiating infrared light to a biological sample provided in the dark room and held in the dark room;
An image acquisition unit that acquires a bright field image from the biological sample in an illumination state in which the infrared light is irradiated on the biological sample, and acquires a weak light image of the biological sample in a non-illuminated state;
A control device having a display unit that superimposes and displays the bright field image and the weak light image;
An image acquisition apparatus comprising: A dark room,
A light source that is provided in the dark room and irradiates a weak infrared light to a biological sample held in the dark room;
An image acquisition unit for acquiring a bright field image including self-luminous light of the biological sample from the biological sample irradiated with the weak infrared light;
An image acquisition apparatus comprising:   The image acquisition apparatus according to claim 2, wherein the weak infrared light is weak so that a background luminance is 1.25 times or more darker than the self-luminous luminance of the biological sample.   The light source emits the weak infrared light by irradiating the biological sample with infrared light during a part of the exposure time when acquiring a bright field image including self-luminous light of the biological sample. The image acquisition device according to claim 3 to be realized.   The image acquisition apparatus according to claim 1, wherein the infrared light is near infrared light. Irradiate a biological sample with infrared light to obtain a bright field image of the biological sample,
Obtaining a faint light image of the biological sample;
A method in which the bright field image and the weak light image are superimposed and displayed.   A method of acquiring a bright field image including self-luminous light of the biological sample by irradiating the biological sample with weak infrared light.   The method according to claim 7, wherein the weak infrared light is weak so that a background brightness is 1.25 times or more darker than the self-luminous brightness of the biological sample.   The weak infrared light is realized by irradiating the biological sample with infrared light during a part of the exposure time when acquiring a bright field image including self-luminous light of the biological sample. 9. The method according to 8.   The method according to claim 6, wherein the infrared light is near infrared light.