WO2017109983A1 - Analysis method and analysis system for faint light-emitting sample - Google Patents

Abstract

This analysis method for a faint light-emitting sample includes: an image acquisition process (S101) of acquiring a faint light image of a faint light-emitting sample as a three-dimensional sample including a plurality of objects to be analyzed which are prepared to emit a faint light; and an analysis process (S102) of analyzing the three-dimensional sample on the basis of the acquired faint light image. The image acquisition process (S101) includes repeatedly determining the opening size of an optical diaphragm inserted in an optical system for acquiring the faint light image on the basis of a predetermined condition (S203), and acquiring the faint light image concerning the three-dimensional sample using the optical diaphragm with the determined opening size (S205).

Description

Method and system for analyzing weakly luminescent sample

The present invention relates to an analysis method and an analysis system for a weakly luminescent sample.

A thick sample (three-dimensional sample) containing a large number of cells to be analyzed, such as embryoid bodies, animal tissues, organs, etc., in a three-dimensional direction. When observing using a 3D image, a three-dimensional image may be reconstructed. A three-dimensional image is reconstructed by capturing a plurality of two-dimensional images while moving the focusing position in the optical axis direction and stacking these images. The three-dimensional image obtained in this way has three-dimensional information and can be used effectively for analysis of a three-dimensional sample.

For example, Japanese Patent No. 5424528 discloses the following technology. That is, in this technique, the analysis target is a sample having a thickness such as a live embryo or tissue that generates bioluminescence as weak light. This sample is provided with a plurality of measurement target parts. In this technique, a thick sample is regarded as a solid, a weak light signal is acquired from a different angle for each measurement target site, and analysis on each measurement site is performed using the signal. The faint light signal acquired from different angles includes certain depth information from the surface of the thick sample, and includes separate three-dimensional information for each measurement site.

Further, for example, Japanese Patent Application Laid-Open No. 2014-119762 discloses a technology related to the following system. That is, this system is a system for analyzing cells and the like. This system can capture bright field images and luminescent or fluorescent images while changing the focus position.

In order to increase the resolution of the obtained 3D image, it is better that the number of 2D images used for 3D reconstruction is large. Further, the depth of focus of the two-dimensional image should be reasonably shallow according to the number of two-dimensional images.

On the other hand, in order to reconstruct a three-dimensional image, an electric system for moving the focal position along the optical axis is necessary. In addition, it takes time to capture a plurality of images for three-dimensional reconstruction. In addition, in a three-dimensional image, it may be difficult to analyze data including setting a region of interest (ROI: Region Of Interest) three-dimensionally. In particular, when acquiring a three-dimensional image of weak light, the time required for photographing tends to be long. For this reason, particularly in the analysis of a three-dimensional sample that generates faint light, there is a desire to accumulate a large amount of information of a three-dimensional sample in one two-dimensional image. The two-dimensional image required for such a reason preferably has a certain depth of focus. Thus, there are cases where both a three-dimensional image and a two-dimensional image are required. In addition, the preferred depth of focus may differ between acquisition of a three-dimensional image and acquisition of a two-dimensional image.

The present invention provides an analysis method and an analysis system for a weakly luminescent sample in which an image with an adjusted depth of focus is acquired in an analysis of a three-dimensional sample having a thickness prepared so as to emit weak light. With the goal.

According to one aspect of the present invention, a method for analyzing a weakly luminescent sample acquires a weak light image of a weakly luminescent sample as a three-dimensional sample prepared to emit weak light and including a plurality of analysis objects. An image acquisition process; and an analysis process for analyzing the three-dimensional sample based on the acquired weak light image, wherein the image acquisition process acquires the weak light image based on a predetermined condition. Repeatedly determining an aperture diameter of the optical aperture inserted into the optical aperture and acquiring a weak light image of the three-dimensional sample using the optical aperture of the determined aperture diameter. In at least one of the repeated image acquisition processes, the aperture diameter includes the analysis target existing at a position different in the optical axis direction of the optical system within the depth of focus. It is determined to be a value.

According to an aspect of the present invention, an analysis system includes an objective optical system, an optical aperture provided in the objective optical system, and an aperture determination unit that determines an aperture diameter of the optical aperture based on a predetermined condition. ,
An aperture drive unit for setting the aperture diameter of the optical aperture to a determined value, and a plurality of analysis objects prepared to emit weak light in a state where the optical aperture is the determined aperture diameter And an imaging device that captures a weak light image of the weak luminescent sample as a three-dimensional sample through the objective optical system.

According to the present invention, there is provided an analysis method and an analysis system for a weakly luminescent sample in which an image with an adjusted focal depth is acquired in an analysis of a three-dimensional sample having a thickness prepared so as to emit weak light. it can.

FIG. 1 is a diagram illustrating an outline of a configuration example of an analysis system according to an embodiment. FIG. 2 is a schematic diagram for explaining the relationship between the aperture diameter of the diaphragm and the obtained image. FIG. 3 is a flowchart illustrating an outline of an example of the analysis method according to the embodiment. FIG. 4 is a flowchart illustrating an outline of an example of an image acquisition process according to an embodiment. FIG. 5 is a diagram for explaining an example of shooting timing according to the first mode. FIG. 6 is a flowchart illustrating an outline of an example of the opening diameter determination process according to the first mode. FIG. 7 is a flowchart illustrating an outline of an example of image generation processing according to the first mode. FIG. 8 is a diagram for explaining an example of shooting timing according to the second mode. FIG. 9 is a flowchart illustrating an outline of an example of the opening diameter determination process according to the third mode. FIG. 10 is a flowchart illustrating an outline of an example of analysis processing according to an embodiment. FIG. 11 is a diagram illustrating an outline of a configuration example of an analysis system according to a modification of the embodiment. FIG. 12 is a diagram illustrating an example of an observation result of a triangular pyramid made of graph paper. FIG. 13 is a diagram illustrating an example of observation results of porous silica beads. FIG. 14 is a diagram showing an example of observation results of femurs of mice knocked in with the clock gene Per2 :: luciferase.

<Outline of analysis>
An embodiment of the present invention will be described. The analysis system according to the present embodiment can be used for analysis of a three-dimensional sample having a thickness prepared so as to emit faint light. Examples of such three-dimensional samples include small animal organs and organs such as mice, embryoid bodies, spheroids, gels, or three-dimensional cell samples cultured in a carrier.

The analysis system according to the present embodiment includes a microscope for acquiring an image. This analysis system can acquire a two-dimensional image of a sample. That is, a two-dimensional weak light image can be acquired for a three-dimensional sample that emits weak light. In addition, the analysis system can acquire a plurality of two-dimensional images for a plurality of different focal planes. This analysis system can generate a three-dimensional image by synthesizing at least one set of two-dimensional images including a plurality of obtained two-dimensional images. That is, a three-dimensional weak light image of a three-dimensional sample that emits weak light can be acquired. In this analysis system, an analysis is performed based on a weak light image such as a two-dimensional image or a three-dimensional image generated in this way. In the analysis, for example, the luminance in the image is obtained.

Further, in the present analysis system, an optical aperture having a variable aperture diameter is provided in an optical system of a microscope such as an objective lens portion. By changing the aperture diameter of this stop, the depth of focus changes. When acquiring a two-dimensional image or a three-dimensional image, the analysis system according to the present embodiment adjusts the aperture diameter of the diaphragm and adjusts the depth of focus of the obtained image.

<System configuration>
A configuration example of the analysis system according to the present embodiment will be described with reference to FIG. The analysis system 1 includes an observation apparatus 100 for observing the sample 900 that is the above-described three-dimensional sample, and a data processing apparatus 200 that controls the operation of the observation apparatus 100 and performs image processing. Furthermore, the analysis system 1 includes a display device 310 for displaying an image processed by the data processing device 200 and an input device 320 used when a user inputs a command to the data processing device 200. The observation apparatus 100 is an apparatus that can acquire a bright field image while illuminating the sample 900, or can acquire a light emission image of the sample 900 under dark conditions. When the observation apparatus 100 and the data processing apparatus 200 cooperate, the sample 900 can be observed.

The observation apparatus 100 includes a stage 110 on which a container 910 containing a sample 900 is placed, a photographing unit 130 for photographing the sample 900, and an illumination unit 160 for illuminating the sample 900.

The container 910 stores the sample 900. As the container 910, a petri dish, a glass slide, a microplate, a gel support, a fine particle carrier, and the like can be used.

In this embodiment, an upright light emitting microscope is used for the photographing unit 130. The photographing unit 130 includes an objective lens 131, an optical aperture 132, an imaging lens 133, and an imaging device 134. The objective lens 131, the optical aperture 132, the imaging lens 133, and the like form part of the objective optical system.

The imaging device 134 captures a light emission image or a bright field image of the sample 900. The imaging device 134 has a solid-state imaging device such as a CCD image sensor or a CMOS image sensor. The imaging device 134 generates image data by photoelectrically converting an image formed on the imaging surface of the solid-state imaging device. The imaging device 134 outputs the generated image data to the data processing device 200. As the imaging device 134, for example, a cooled CCD camera can be used. As the cooling CCD, for example, a cooling CCD of 0 ° C. or lower can be used. As the cooling CCD, a cooling CCD of preferably −80 ° C. to −30 ° C., particularly a cooling CCD of about −60 ° C. can be used.

The objective lens 131 and the imaging lens 133 form an image of the sample 900 on the imaging surface of the imaging device of the imaging device 134. The diaphragm 132 is provided behind the objective lens 131. The diaphragm 132 is configured such that the opening diameter can be changed. The diaphragm 132 may be inserted at another position in the photographing unit 130.

The observation apparatus 100 is provided with a diaphragm driving unit 142 that changes the aperture diameter of the diaphragm 132 under the control of the data processing apparatus 200. Further, the photographing unit 130 is provided with a lens moving mechanism 136 that moves the objective lens 131 along its optical axis (Z axis) in order to change the focal plane. The observation apparatus 100 is provided with an objective lens driving unit 144 that operates the lens moving mechanism 136 under the control of the data processing apparatus 200.

The stage 110 can move in a plane perpendicular to the optical axis of the photographing unit 130 (in the XY plane). The observation apparatus 100 is provided with a stage drive unit 120 that moves the stage 110 under the control of the data processing apparatus 200. The stage drive unit 120 moves the stage 110 two-dimensionally.

The illumination unit 160 irradiates the sample 900 with light for bright field observation (for example, white light). The illumination unit 160 includes a light source 161, a shutter 162, and an illumination optical system 163. The light source 161 includes a halogen lamp that emits light for bright field observation.

The shutter 162 is a shutter that switches whether the sample 900 is irradiated with light for bright field observation. The observation apparatus 100 is provided with a shutter driving unit 170 that drives the shutter 162 under the control of the data processing apparatus 200.

The illumination optical system 163 includes a collector lens 164 and an illumination fiber 165. The incident end of the illumination fiber 165 is provided at the condensing position of the collector lens 164. The exit end of the illumination fiber 165 is on the objective lens 131 side of the stage 110 (above the sample 900 in the illustrated upright light emission microscope), and the exit direction of the illumination light is the direction of the sample 900 (or the container 910). Less than 90 degrees with respect to the optical axis of the objective lens 131, preferably 30 to 65 degrees so that the optical axis is inclined toward the vicinity of the center of the bottom surface and the incidence on the objective lens 131 is minimized. It is provided with an acute angle inclination.

The light from the light source 161 condensed by the collector lens 164 is emitted from the exit end of the illumination fiber 165 at a predetermined angle, so that the sample 900 can be irradiated almost uniformly from the objective lens 131 side. it can. In this way, by illuminating from the objective lens 131 side, a bright field image with good line of sight can be obtained without causing a shadow for a thick sample.

The data processing device 200 includes an arithmetic circuit 210 and a storage device 280. The arithmetic circuit 210 is a circuit that performs various calculations. The storage device 280 includes a storage device that stores various programs and various parameters used in the arithmetic circuit 210. The storage device 280 stores information such as the obtained image and analysis results. The storage device 280 may include a RAM that temporarily stores information used when the arithmetic circuit 210 performs an operation. The storage device 280 can include, for example, a semiconductor memory, a hard disk, various ROMs, and the like.

FIG. 1 shows functional blocks of the arithmetic circuit 210. That is, the arithmetic circuit 210 includes an imaging control unit 222, an aperture determination unit 224, an imaging device control unit 232, an aperture control unit 234, a position control unit 236, a shutter control unit 238, an image processing unit 242, The image synthesizing unit 244, the image analyzing unit 246, and the display control unit 248 are provided.

The imaging control unit 222 controls overall operations such as an operation related to image acquisition using the observation apparatus 100, an operation related to display of the acquired image, and an analysis based on the acquired image.

The aperture determination unit 224 determines the aperture diameter of the aperture 132 based on the information acquired from the imaging control unit 222. The aperture determination unit 224 transmits the determined aperture diameter to the aperture control unit 234.

The aperture control unit 234 controls the operation of the aperture 132 so that the aperture diameter of the aperture 132 becomes the aperture diameter acquired from the aperture determination unit 224 under the instruction of the imaging control unit 222. That is, the aperture controller 234 outputs a control signal to the aperture driver 142.

The imaging device control unit 232 controls the operation of the imaging device 134 under the instruction of the imaging control unit 222. The imaging device control unit 232 controls, for example, imaging timing, exposure time, and the like.

The position control unit 236 controls the position of the objective lens 131 and the position of the stage 110 under the instruction of the imaging control unit 222. That is, the position control unit 236 outputs a control signal to the objective lens driving unit 144 and the stage driving unit 120.

The shutter control unit 238 controls the operation of the shutter 162 under the instruction of the shooting control unit 222. That is, the shutter control unit 238 outputs a control signal to the shutter driving unit 170. For example, the shutter control unit 238 opens the shutter 162 when acquiring a bright field image, and closes the shutter 162 when acquiring a light emission image.

The image processing unit 242 acquires image data from the imaging device 134 under the control of the imaging control unit 222 and performs image processing on the image data. For example, the image processing unit 242 creates two-dimensional image data. The image processing unit 242 transmits the image data after the image processing to the image synthesis unit 244, the image analysis unit 246, or the display control unit 248. In addition, the image processing unit 242 causes the storage device 280 to record the processed image.

The image composition unit 244 synthesizes a three-dimensional image based on the two-dimensional image acquired from the image processing unit 242 under the control of the imaging control unit 222. The image composition unit 244 transmits the created three-dimensional image to the image analysis unit 246 or the display control unit 248. In addition, the image composition unit 244 records the created three-dimensional image in the storage device 280.

The image analysis unit 246 performs analysis using the two-dimensional image acquired from the image processing unit 242 or the three-dimensional image acquired from the image composition unit 244 under the control of the imaging control unit 222. For example, the image analysis unit 246 calculates the light emission amount of the three-dimensional sample based on the image data. The image analysis unit 246 transmits the analysis result to the display control unit 248 in a necessary format as necessary. In addition, the image analysis unit 246 records the analysis result in the storage device 280.

The display control unit 248 causes the display device 310 to display an image based on information acquired from the image processing unit 242, the image synthesis unit 244, or the image analysis unit 246 under the control of the imaging control unit 222.

The arithmetic circuit 210 includes an integrated circuit such as a Central Processing Unit (CPU), an Application Specific Integrated Circuit (ASIC), or a Field Programmable Gate Array (FPGA). The arithmetic circuit 210 may be configured by one integrated circuit or the like, or may be configured by combining a plurality of integrated circuits or the like provided for each functional block, for example. The operations of these integrated circuits are performed in accordance with, for example, a program recorded in the storage device 280 or a recording area in the integrated circuit.

The display device 310 is a general display device such as a liquid crystal display or an organic EL display. The display device 310 displays various images under the control of the display control unit 248. In addition to the display device 310, for example, a printer that prints display content on paper may be provided.

The input device 320 is a general input device such as a keyboard, a mouse, a touch panel, and a switch. The input device 320 transmits input from the user to the data processing device 200.

<Three-dimensional sample>
Examples of the three-dimensional sample include a small animal organ or organ such as a mouse, a living tissue, an embryoid body, a spheroid, or a three-dimensional cell sample cultured in a gel or a carrier as described above. The present embodiment includes obtaining a light emission image of a three-dimensional sample that emits weak light. For this reason, for example, a photoprotein gene is introduced into the cells constituting the sample. As the photoprotein, for example, luciferase can be used. When luciferase is used as the photoprotein, luciferin, which is a luminescent substrate, is introduced into the sample. In general, a cell into which a photoprotein gene has been introduced only emits bioluminescence with an extremely weak luminance of 1/100 or less as compared with a fluorescent protein, and it is difficult to image at a video rate. Nevertheless, photoproteins produce bioluminescence that is highly correlated with the level of gene expression in the cell, so by measuring faint light over a long period of time, we can accurately capture weak changes in expression level. be able to. Such faint light that makes imaging at a video rate difficult is very suitable for quantifying faint biological activity. However, a microscope-based luminescence imaging system (luminescence microscope) that can acquire faint light such as bioluminescence with high sensitivity is in the process of development, and neither a technique nor an apparatus that is applied to various biological activities has been studied.

When investigating the strength of gene expression, which is an example of biological activity, using luciferase activity as an index, luciferase is introduced into a living cell as a reporter gene. In this case, when the target DNA fragment is connected upstream or downstream of the luciferase gene, the influence of the DNA fragment on transcription can be examined in time series. In addition, by connecting a gene such as a transcription factor that is thought to affect transcription to an expression vector and co-expressing it with the reporter gene, the effect of the gene product on the expression of the reporter gene can be examined.

<Relationship between aperture diameter and resulting image>
The relationship between the aperture diameter of the aperture 132 and the image obtained by the imaging device 134 will be described with reference to the schematic diagram shown in FIG. In FIG. 2, the left column shows a case where a two-dimensional image is obtained, and the right column shows a case where a three-dimensional image is obtained. In FIG. 2, the upper stage shows a case where the aperture diameter of the diaphragm 132 is large, and the lower stage shows a case where the aperture diameter of the diaphragm 132 is small.

The upper figure in each column shows the plane including the optical axis. That is, these drawings show schematic views of the sample 900 on the bottom surface of the container 910 as viewed from the side. Each white rectangle 810 in the drawing schematically shows a region where an image can be obtained by one imaging. That is, the height of each rectangle 810 schematically indicates the depth of focus. In the case of acquiring a two-dimensional image, one two-dimensional image is acquired, and thus one rectangle 810 is shown. On the other hand, in the case of acquiring a three-dimensional image, a plurality of two-dimensional images are obtained while moving the objective lens 131 along its optical axis by the lens moving mechanism 136, and thus a plurality of rectangles 810 are shown.

The lower figure in the 2D image column schematically shows the obtained 2D image 820. These two-dimensional images 820 are images representing a plane perpendicular to the optical axis, and are images representing a cross section of the sample 900 related to the region indicated by the rectangle 810. The lower figure in the three-dimensional image column schematically shows a cross-sectional image 830 for a plane including the optical axis of the obtained three-dimensional image.

As shown in FIG. 2, when the aperture diameter of the diaphragm 132 is large, the depth of focus is shallow. For this reason, the obtained two-dimensional image 820 includes information on the sample in a narrow range related to the in-focus plane, and does not include information on the sample in the area outside the in-focus plane. For example, the first structure 901 in the sample 900 is represented in the two-dimensional image 820, but the second structure 902 is not represented in the two-dimensional image 820. On the other hand, when the aperture diameter of the stop 132 is small, the depth of focus is deep. For this reason, light in a wide area in the depth direction is accumulated in one two-dimensional image. As a result, the obtained two-dimensional image includes information on a wide range of samples including a region deviated from the focal plane. For example, the first structure 901 and the second structure 902 in the sample 900 are both represented in the two-dimensional image 820. Therefore, in order to obtain a large amount of information with one two-dimensional image, it is preferable that the aperture diameter of the diaphragm 132 is small. In order to obtain a high-resolution image with a single two-dimensional image, it is preferable that the aperture diameter of the diaphragm 132 is large.

As for the three-dimensional image, as shown in FIG. 2, the higher the aperture diameter of the diaphragm 132, the higher the resolution. On the other hand, if the aperture diameter of the stop 132 is small, the observation regions overlap and the resolution in the depth direction is deteriorated, and the obtained three-dimensional image has a poor resolution. Therefore, when obtaining a three-dimensional image, it is preferable that the aperture diameter of the diaphragm 132 is large.

In the analysis system according to the present embodiment, when acquisition of a two-dimensional image having a lot of information in the depth direction is required, the aperture diameter of the diaphragm 132 is reduced and the two-dimensional image is recorded in a state where the focal depth is deep. Acquisition is performed. On the other hand, when acquisition of a three-dimensional image with high resolution is required, the aperture diameter of the diaphragm 132 is increased and a plurality of two-dimensional images are acquired with a shallow depth of focus. By synthesizing the two-dimensional image having the shallow depth of focus thus obtained, one high-resolution three-dimensional image can be obtained.

It should be noted that there are various uses for 2D images and 3D images. For example, according to a two-dimensional image, analysis and visual recognition are easy. For this reason, a two-dimensional image can be used for grasping the sample outline. On the other hand, when detailed analysis is required, a three-dimensional image can be used. In addition, based on the acquisition of the two-dimensional image, when a predetermined change is detected in the two-dimensional image, the necessary information can be acquired efficiently by acquiring the three-dimensional image.

<analysis method>
A method for analyzing a weakly luminescent sample having three dimensions according to the present embodiment will be described. As shown in FIG. 3, the analysis method includes an image acquisition process in step S101 and an analysis process in step S102. In the image acquisition process of step S101, an image of the sample used for analysis is acquired by repeatedly executing a predetermined operation. In the analysis processing in step S102, the sample is analyzed based on the obtained image. Note that acquisition of an image and analysis of the obtained image may be alternately repeated.

The image acquisition process will be described. In the image acquisition process, a plurality of images including a two-dimensional image or a three-dimensional image are acquired over time. That is, time-lapse observation is performed. This image acquisition may be controlled manually or may be controlled by the data processing device 200, for example. An example of the image acquisition process controlled by the data processing device 200 will be described with reference to FIG.

In step S201, the imaging control unit 222 of the data processing device 200 performs various settings related to image acquisition. The data processing apparatus 200 sets a series of shooting time timings, the number of images to be acquired, a focus position, and the like based on values input by the user, for example.

In step S202, the shooting control unit 222 of the data processing device 200 determines whether it is time to perform shooting. For example, the imaging control unit 222 measures the elapsed time from the start of imaging. In step S202, the shooting control unit 222 compares the elapsed time from the start of shooting with the timing of shooting set in step S201, and determines whether to perform shooting. When it is not time to perform shooting, the process proceeds to step S206, and the processes of steps S203 to S205 are skipped. On the other hand, when it is time to shoot, the process proceeds to step S203.

In step S203, the aperture determining unit 224 of the data processing device 200 performs an aperture diameter determining process for determining the aperture diameter of the diaphragm 132 based on a predetermined condition. In this embodiment, when a two-dimensional image is acquired, a small aperture diameter with a deep focal depth is selected. On the other hand, when a three-dimensional image is acquired, a large aperture diameter with a shallow depth of focus is selected. The small opening diameter and the large opening diameter may be determined in advance according to the optical conditions of the observation apparatus 100, respectively. For example, the small opening diameter can be determined such as how many mm the diameter of the aperture of the diaphragm 132 is, and the large aperture diameter can be determined such as how many mm the diameter of the aperture of the diaphragm 132 is. Alternatively, the small opening diameter and the large opening diameter may be respectively designated by the user in the setting in step S201.

In step S204, the aperture control unit 234 of the data processing device 200 controls the operation of the aperture 132 so that the determined aperture diameter is obtained.

In step S205, the data processing apparatus 200 performs image generation processing for capturing an image and generating a two-dimensional image or a three-dimensional image. That is, when a small aperture diameter is selected in step S203 and the aperture diameter of the diaphragm 132 is set to be small in step S204, one two-dimensional image is acquired. On the other hand, when a large aperture diameter is selected in step S203 and the aperture diameter of the diaphragm 132 is set to be large in step S204, a plurality of two-dimensional images are acquired, and a three-dimensional image is obtained based on these two-dimensional images. An image is created.

In step S206, the imaging control unit 222 of the data processing device 200 determines whether or not a series of imaging has been completed. When it is determined that a series of shooting has not been completed, the process returns to step S202. In this way, the set number of images is acquired for the period set in step S201. When a series of photographing is finished, the image acquisition process is finished.

<Image acquisition mode>
In the analysis system 1 according to the present embodiment, three operation modes for image acquisition are prepared. In any mode, in at least one of the repeated image acquisitions, the aperture diameter is set to a value that includes the analysis target at different positions in the optical axis direction of the objective optical system within the depth of focus. It is determined. These modes will be described in order.

(First mode)
The first mode is a mode in which a two-dimensional image and a three-dimensional image are acquired at the image acquisition timing set in step S201, respectively. The timing at which an image is acquired in the first mode will be described with reference to FIG. In the first mode, the analysis system 1 first reduces the aperture diameter of the diaphragm 132 to a state where the depth of focus is deep, and in this state, captures one image and acquires one two-dimensional image. Subsequently, the analysis system 1 increases the aperture diameter of the diaphragm 132 so that the focal depth is shallow, and in this state, the analysis system 1 performs a plurality of times along the depth direction of the weakly luminescent sample (a direction substantially perpendicular to the two-dimensional image). Are taken, and the obtained images are combined to obtain a single three-dimensional image. The analysis system 1 repeatedly performs such image acquisition as a set of the two-dimensional image and the three-dimensional image at the timing set in step S201. In order to obtain a predetermined exposure amount, when the aperture diameter is small, it is necessary to expose for a longer time than when the aperture diameter is large.

In this way, by obtaining an image as a set of a two-dimensional image and a three-dimensional image using different aperture diameters of optical stops, image information in the two-dimensional image is obtained based on the three-dimensional image. Can be easily associated three-dimensionally. Therefore, as the best mode in the first mode, a three-dimensional analysis of weak light can be performed by acquiring a minimum three-dimensional image by repeatedly acquiring a two-dimensional image provided with three-dimensional information by a three-dimensional image. It can be executed continuously.

In the first mode, for example, the opening diameter is changed to a small state immediately before shooting for a two-dimensional image, and the opening diameter is set to a large state immediately before the first shooting for a three-dimensional image is performed. Be changed. Alternatively, for example, the aperture diameter is changed to a large state immediately after shooting for a two-dimensional image, and the aperture diameter is changed to a small state immediately after the last shooting for a three-dimensional image is performed. The

In the first mode, the opening diameter determination process performed in step S203 will be described with reference to FIG. In step S301, the opening determination unit 224 of the data processing device 200 determines whether it is time to change the opening diameter to a small state. For example, in a setting in which the aperture diameter is changed to a small state immediately before shooting for a two-dimensional image and the aperture diameter is changed to a large state immediately before the first shooting for a three-dimensional image is performed, 2 is set. It is determined whether or not it is immediately before photographing for a three-dimensional image is performed. Alternatively, for example, the setting is made such that the aperture diameter is changed to a large state immediately after shooting for a two-dimensional image and the aperture diameter is changed to a small state immediately after the last shooting for a three-dimensional image is performed. It is determined whether or not it is immediately after the last shooting for a three-dimensional image is performed. When it is time to reduce the opening diameter, the process proceeds to step S302. In step S302, the aperture determining unit 224 determines to change the aperture diameter of the aperture 132 to a small state. Thereafter, the opening diameter determination process ends, and the process returns to the image acquisition process described with reference to FIG.

If it is determined in step S301 that it is not time to change the aperture diameter to a small state, the process proceeds to step S303. In step S303, the opening determination unit 224 determines whether it is time to change the opening diameter to a larger state. For example, in a setting in which the aperture diameter is changed to a small state immediately before shooting for a two-dimensional image and the aperture diameter is changed to a large state immediately before the first shooting for a three-dimensional image is performed, 3 It is determined whether or not it is just before the first shooting for a three-dimensional image is performed. Alternatively, for example, the setting is made such that the aperture diameter is changed to a large state immediately after shooting for a two-dimensional image and the aperture diameter is changed to a small state immediately after the last shooting for a three-dimensional image is performed. In, it is determined whether or not it is immediately after shooting for a two-dimensional image. When it is time to change the opening diameter to a larger state, the process proceeds to step S304. In step S304, the aperture determining unit 224 determines to change the aperture diameter of the aperture 132 to a large state. Thereafter, the opening diameter determination process ends, and the process returns to the image acquisition process described with reference to FIG.

If it is determined in step S303 that it is not time to change the aperture diameter to a larger state, the process proceeds to step S305. In step S305, the opening determination unit 224 determines to maintain the opening diameter. Thereafter, the opening diameter determination process ends, and the process returns to the image acquisition process described with reference to FIG.

After the aperture diameter determination process, in step S204, the aperture control unit 234 of the data processing device 200 controls the operation of the aperture 132 so that the determined aperture diameter is obtained. For example, when it is determined in the opening diameter determination process in step S203 that the opening diameter is changed to a small state, the diaphragm 132 is changed to a state where the opening diameter is small. On the other hand, when it is determined to change the aperture diameter to a large state, the diaphragm 132 is changed to a state where the aperture diameter is large. Further, when it is determined to maintain the aperture diameter, the diaphragm 132 is not changed.

Next, the image generation process performed in step S205 will be described with reference to FIG.

In step S401, the imaging control unit 222 of the data processing device 200 determines whether or not the aperture diameter of the diaphragm 132 determined in step S203 and set in step S204 is small. When the opening diameter is small, the process proceeds to step S402. In step S <b> 402, the imaging control unit 222 causes the imaging device 134 to perform imaging at a predetermined focal plane once via the imaging device control unit 232, and acquires image data related to one two-dimensional image. The focal plane set here may be a focal plane designated in advance by the user in step S201, for example. Further, for example, the focal plane may include a region with the highest luminance in the previously acquired three-dimensional image.

In step S403, the imaging control unit 222 causes the image processing unit 242 to create one two-dimensional image based on the image data acquired in step S402. Thereafter, the image generation process ends, and the process returns to the image acquisition process described with reference to FIG.

If it is determined in step S401 that the aperture diameter of the diaphragm 132 is not small, that is, the aperture diameter of the diaphragm 132 is large, the process proceeds to step S404. In step S <b> 404, the imaging control unit 222 causes the imaging device 134 to repeatedly perform imaging while changing the focal plane by moving, for example, the stage 110 in the optical axis direction via the position control unit 236. Image data relating to a three-dimensional image is acquired. In step S405, the imaging control unit 222 causes the image composition unit 244 to perform a reconstruction process for creating one three-dimensional image based on the image data of the plurality of two-dimensional images acquired in step S404. Thereafter, the image generation process ends, and the process returns to the image acquisition process described with reference to FIG.

At the timing adjusted in step S202, the two-dimensional image and the three-dimensional image are repeatedly acquired by the processing in steps S203 to S205, whereby the two-dimensional image and the three-dimensional image are repeatedly acquired as a set. It will be. Although an example in which a 3D image is acquired after a 2D image is shown here, a 3D image may be acquired first and then a 2D image may be acquired.

(Second mode)
An outline of the second mode will be described with reference to FIG. In the second mode, as shown in FIG. 8, photographing for obtaining a two-dimensional image is repeatedly performed in a state where the aperture diameter with a large depth of focus is small, and photographing with a large aperture diameter with a small depth of focus is performed at a predetermined frequency. One three-dimensional image acquisition is performed by performing it several times. For example, the exposure time required for acquiring a two-dimensional image is, for example, about 10 minutes, and for acquiring a three-dimensional image, for example, the time required for acquiring one two-dimensional image is, for example, about three minutes. Such a two-dimensional image acquisition is repeated 10 times.

In the second mode, the analysis can be performed mainly based on the information included in the two-dimensional image, for example, when the cell moves in the living tissue, and the information is partially included in the three-dimensional image. This is effective when it is necessary to perform an analysis based on this. That is, three-dimensional analysis using a two-dimensional image can be continuously performed while accurately reflecting information based on the three-dimensional image.

Also in the second mode, for example, the opening diameter is changed to a large state immediately before the first shooting for the three-dimensional image is performed, and the opening diameter is small immediately before the shooting for the two-dimensional image is performed. Changed to

Also in the second mode, the opening diameter determination process performed in step S203 is performed according to the procedure described with reference to the flowchart of FIG. That is, in step S301, the opening determination unit 224 determines whether it is time to change the opening diameter to a small state. For example, it is determined whether or not it is immediately before photographing for a two-dimensional image is performed. When it is just before photographing for a two-dimensional image is performed, in step S302, the aperture determination unit 224 determines to make the aperture diameter of the aperture 132 small. In step S303, the opening determination unit 224 determines whether it is time to change the opening diameter to a larger state. For example, it is determined whether it is immediately before the first shooting for a three-dimensional image is performed. When it is just before the first shooting for a three-dimensional image is performed, in step S304, the aperture determination unit 224 determines to increase the aperture diameter of the aperture 132.

Also in the second mode, the image generation process performed in step S205 is performed according to the procedure described with reference to the flowchart of FIG. That is, when the aperture diameter of the diaphragm 132 is small, one image data is acquired in step S402, and one two-dimensional image is created in step S403. On the other hand, when the aperture diameter of the diaphragm 132 is large, a plurality of image data is acquired in step S404, and one three-dimensional image is created in step S405.

In the second mode, a two-dimensional image or a three-dimensional image is repeatedly acquired at the timing adjusted in step S202 by the processing in steps S203 to S205.

As described above, when the first opening diameter is a small opening diameter and the second opening diameter is a large opening diameter, the opening diameter of the diaphragm 132 is set to the first opening diameter at the repeated first timing. The aperture diameter of the diaphragm 132 is set to the second aperture diameter at the second timing repeated. When the aperture diameter of the diaphragm 132 is the second aperture diameter, a set of two-dimensional weak light images for a plurality of different focal planes are acquired, and the set of two-dimensional weak light images are obtained. A three-dimensional weak light image is generated by combining the images.

(Third mode)
The third mode will be described. In the third mode, first, a two-dimensional image is repeatedly acquired. The obtained two-dimensional image is sequentially analyzed. When a change larger than a predetermined change is detected in the two-dimensional image, acquisition of the three-dimensional image is performed. For example, for images of luminescence as faint light (light generated from cells by the luciferase gene), there was a change in luminescence intensity that could be interpreted as a significant change in biological activity (eg gene expression level). In such a case, the analysis system 1 can be configured such that a three-dimensional image is acquired assuming that a large change is detected. Alternatively, the analysis system 1 can be configured so that a change is detected when the emission intensity that is recognized as having biological activity is set as a threshold and the emission intensity exceeds the threshold. Further, the obtained three-dimensional image is sequentially analyzed. Then, when it is detected that the change is smaller than the predetermined change in the three-dimensional image, acquisition of the two-dimensional image is performed.

In the third mode, in the setting performed in step S201, the imaging condition in a state where the aperture diameter is small, that is, the depth of focus is deep is set as the initial value of the aperture diameter of the diaphragm 132.

The opening diameter determination process performed in step S203 in the third mode will be described with reference to FIG. In the image generation process performed in step S205, the same process as the process described with reference to FIG. 7 is performed. In step S501, the opening determination unit 224 of the data processing device 200 determines whether or not the opening diameter is small. When the opening diameter is small, the process proceeds to step S502. In step S502, the image analysis unit 246 analyzes a two-dimensional image obtained in the past, and the aperture determination unit 224 determines whether a predetermined change is detected in the two-dimensional image.

When the change is detected in step S502, for example, when the change in luminance in the two-dimensional image obtained by two successive acquisitions is greater than a predetermined value, the luminance value in the obtained two-dimensional image May exceed the predetermined threshold value, the luminance value in the obtained two-dimensional image may exceed the threshold value corresponding to the predetermined upper limit value, and / or the case may be below the threshold value corresponding to the predetermined lower limit value. . Here, a three-dimensional analysis corresponding to various changes in weak light can be performed by applying a range of change amounts composed of threshold values of both an upper limit value and a lower limit value. If the threshold value corresponding to the lower limit value is not reached, by increasing the aperture diameter of the optical stop, the amount of light collection can be increased and the limit of analysis can be expanded even if the luminance value becomes extremely weak. In this example, since the initial value of the aperture diameter of the diaphragm 132 is set to a small state, a two-dimensional image is always acquired in the first image acquisition.

When it is determined in step S502 that a change has been detected, the process proceeds to step S503. In step S503, the aperture determining unit 224 determines to change the aperture diameter of the aperture 132 to a large state. Thereafter, the opening diameter determination process ends, and the process returns to the image acquisition process described with reference to FIG. As a result, one three-dimensional image is created in the next image generation process.

When it is determined in step S502 that no change has been detected in the two-dimensional image, the process proceeds to step S504. In step S504, the aperture determining unit 224 determines to maintain the aperture diameter of the diaphragm 132 in a small state. Thereafter, the opening diameter determination process ends, and the process returns to the image acquisition process described with reference to FIG. As a result, one two-dimensional image is created in the next image generation process.

When it is determined in step S501 that the opening diameter is not small, the process proceeds to step S505. In step S505, the image analysis unit 246 analyzes a three-dimensional image obtained in the past, and the aperture determination unit 224 determines whether a predetermined change is detected in the three-dimensional image.

The case where a change is detected in step S505 is, for example, a case where a change in which the luminance is reduced is detected in a three-dimensional image obtained by two successive acquisitions. This may be the case when the value becomes larger than the value, or when the luminance value in the obtained three-dimensional image falls below a predetermined threshold.

When it is determined in step S505 that no change has been detected, the process proceeds to step S506. In step S506, the aperture determination unit 224 determines to change the aperture diameter of the aperture 132 to a small state. Thereafter, the opening diameter determination process ends, and the process returns to the image acquisition process described with reference to FIG. As a result, one two-dimensional image is created in the next image generation process.

When it is determined in step S505 that a change has been detected in the three-dimensional image, the process proceeds to step S507. In step S507, the aperture determining unit 224 determines to maintain the aperture diameter of the diaphragm 132 in a large state. Thereafter, the opening diameter determination process ends, and the process returns to the image acquisition process described with reference to FIG. As a result, one three-dimensional image is created in the next image generation process.

As described above, for example, when the brightness of the weak light in the acquired image is high, a three-dimensional image is created. On the other hand, for example, when the brightness of the weak light in the acquired image is low, a two-dimensional image is created.

In general, when there is a change in luminescence intensity that can be interpreted as a significant change in biological activity as described above, it is desirable to analyze the state of the change in detail using a three-dimensional image. is there. According to the third mode, a necessary three-dimensional image is acquired only when a change occurs, and only a two-dimensional image is acquired when no change occurs. As a result, the amount of data can be suppressed. In addition, according to the two-dimensional image, the data listability is improved. For example, light emission observation is often performed over a long period of time, and if a large amount of three-dimensional images having a large amount of data is recorded, the amount of data becomes enormous, which may hinder analysis. A three-dimensional image is an image that is not suitable for grasping the contents of the image at a glance.

Thus, when the first opening diameter is a small opening diameter and the second opening diameter is a large opening diameter, the initial value of the opening diameter of the diaphragm 132 is set to the first opening diameter. Then, when the weak light images repeatedly acquired with the first opening diameter are compared, and the change in the luminance value of the weak light image becomes larger than a predetermined value, the opening diameter of the diaphragm 132 is the second opening diameter. Set to Further, when the aperture diameter of the diaphragm 132 is set to the second aperture diameter, a set of two-dimensional weak light images for a plurality of different focal planes is acquired, and the set of two-dimensional weak light images is obtained. A three-dimensional weak light image is generated by synthesis.

Here, an example is shown in which the data processing device 200 determines whether or not the brightness of the two-dimensional image has changed. However, it is not limited to this. For example, each time a two-dimensional image is acquired, it is displayed on the display device 310, and the user may input the timing for acquiring the three-dimensional image to the data processing device 200 while confirming this display. In this case, when an instruction to acquire a three-dimensional image is input, the data processing device 200 performs an operation for acquiring a three-dimensional image.

As described above, the operations repeatedly executed in the image acquisition processing in step S101 include predetermined operations such as opening diameter determination processing, setting of the opening diameter of the aperture 132, image generation processing, and the like. By repeatedly performing these operations, an image of the sample used for analysis is acquired. As an example of the operation in each mode described above, an example is shown in which only two states, the state in which the aperture diameter of the diaphragm 132 is large and the state in which it is small, are taken. However, it is not limited to this. You may take a state with three or more opening diameters. For example, when a two-dimensional image is acquired, a plurality of types of two-dimensional images obtained in a plurality of states with different opening diameters may be acquired according to the use of the two-dimensional image, or the opening diameter may be appropriately set. It may be selected. Further, when a three-dimensional image is acquired, the aperture diameter of the diaphragm 132 may be appropriately selected depending on the relationship between the time required for photographing and the resolution of the obtained three-dimensional image.

<About analysis processing>
In the analysis process performed in step S102, various analyzes are performed based on the two-dimensional image or the three-dimensional image acquired in the image acquisition process in step S101. In the analysis process, for example, the luminance distribution in the image is analyzed. In the analysis process, for example, a change in luminance distribution in the image with respect to the elapsed time is analyzed.

In the analysis process, a necessary image may be selected from a plurality of two-dimensional images and three-dimensional images obtained in a series of image acquisitions and analyzed. As an example, when a two-dimensional image and a three-dimensional image are acquired as a set of information as in the first mode shown in FIG. 5, an example in which the selected image is analyzed will be described with reference to FIG. I will explain.

In step S601, the display control unit 248 of the data processing device 200 causes the display device 310 to display a list of acquired two-dimensional images.

In step S602, the imaging control unit 222 of the data processing device 200 determines whether one two-dimensional image is selected from the plurality of displayed two-dimensional images. If a two-dimensional image has not been selected, the process proceeds to step S604. On the other hand, when a two-dimensional image is selected, the process proceeds to step S603. In step S603, the image analysis unit 246 of the data processing device 200 performs analysis using a three-dimensional image corresponding to the selected two-dimensional image. The three-dimensional image corresponding to the selected two-dimensional image is, for example, a three-dimensional weak light image synthesized based on a set of two-dimensional weak light images acquired at the timing closest to the selected two-dimensional image. obtain. Thereafter, the process proceeds to step S604.

In step S604, the imaging control unit 222 determines whether to end the analysis process. If the analysis process is not terminated, the process returns to step S601. On the other hand, when the analysis process ends, the analysis process ends.

In this way, 2D images can be used as thumbnail images. The two-dimensional image according to the present embodiment is an image obtained by increasing the depth of focus by reducing the aperture diameter of the diaphragm 132. For this reason, a lot of information is included in the two-dimensional image. For this reason, the two-dimensional image according to the present embodiment is suitable as a thumbnail image. As a result, the user can quickly select a notable image. This is effective in improving the efficiency of the entire analysis.

<Advantages of this analysis system>
According to the present embodiment, by changing the aperture diameter of the diaphragm 132, it is possible to appropriately acquire a two-dimensional image with a large amount of information and a three-dimensional image with a high resolution for a three-dimensional sample. A two-dimensional image requires a shorter time for photographing than a three-dimensional image. For this reason, according to this embodiment, information can be efficiently collected within a limited time. In addition, the data size of the two-dimensional image is smaller than that of the three-dimensional image. For this reason, according to this embodiment, it becomes easy to manage data by suppressing the generation of unnecessary data. In addition, two-dimensional images are easier to handle data, such as setting ROI, than three-dimensional images. In addition, the two-dimensional image has better listability than the three-dimensional image. On the other hand, a three-dimensional image has a large amount of information. Therefore, acquiring a three-dimensional image as necessary is effective for more detailed sample analysis and three-dimensional grasp of the sample state.

<Modification>
The configuration of the observation apparatus 100 of the above-described embodiment can be changed as appropriate. For example, in the example illustrated in FIG. 1, the sample 900 is irradiated with illumination light from the objective lens 131 side. However, it is not limited to this. Illumination light may be irradiated from the side opposite to the objective lens 131 with the sample 900 interposed therebetween. In this case, for example, a configuration as shown in FIG. 11 can be adopted. That is, the illumination unit 160 is arranged on the opposite side of the sample unit 130 with respect to the sample 900. Instead of the illumination fiber 165 in the example shown in FIG. 1, a condenser lens 167 is provided in the observation apparatus 100. That is, the illumination optical system 163 includes a collector lens 164 and a condenser lens 167. The collector lens 164 and the condenser lens 167 of the illumination optical system 163 collect white light from the light source 161 on the sample 900. In the above-described embodiment, an example in which an upright microscope is used has been described, but the present invention is not limited thereto. For example, an inverted microscope (for example, LUMINOVIEW 200, which is an inverted luminescence imaging system manufactured by Olympus Corporation) may be used.

In the above-described embodiment, the example in which the optical system of the photographing unit 130 is changed by moving the objective lens 131 in order to change the in-focus plane has been described. However, the present invention is not limited to this, and the stage 110 may be moved along the optical axis.

In the above-described embodiment, whether the two-dimensional image and the three-dimensional image are bright-field images or light-emitting images is not limited. Only the luminescent image may be acquired, or the bright field image may be acquired together with the luminescent image. Further, the light emission image and the bright field image may be acquired at predetermined timings, respectively. Further, the acquisition of the luminescent image and the acquisition of the bright field image may be performed according to a predetermined condition.

In the above-described embodiment, an example in which a three-dimensional sample is prepared to emit light and a luminescence image is acquired is shown. However, it is not limited to this. If the three-dimensional sample is prepared so as to emit fluorescence and the observation apparatus 100 is configured to acquire a fluorescence image, a fluorescence image that makes it difficult to acquire a three-dimensional image at a video rate may be acquired. . In this way, in addition to light emission, the weak light emits weak light that is difficult to perform three-dimensional analysis at the video rate. A sample exhibiting such an event corresponds to the three-dimensional analysis of faint light in the present invention because it can be considered that the thickness of the sample is relatively large. Where similar events are indicated, the use of fluorescence (eg, fluorescent proteins) can also be included.

[First embodiment]
<Observation method>
Bright field images were taken using an upright luminescence imaging system. Here, as in the observation apparatus 100 shown in FIG. 1, an upright light emission imaging system having a configuration in which illumination light is irradiated from the objective lens 131 side is used. The objective lens used is an objective lens (XLFLUOR4X / 340; Olympus Corporation) having a magnification of 4 times. In this lens, a sample was photographed in each of a state in which no diaphragm was attached (NA: 0.28) and a diaphragm having a different aperture diameter was attached to the rear of the objective lens, and a two-dimensional image was obtained. There are three types of aperture diameters of the attached diaphragm: φ16.8 mm, φ12.6 mm, and φ8.4 mm. The NA when the opening diameter is φ8.4 mm corresponds to 0.0933. Each exposure time was 50 milliseconds. The following two types of samples were used.

(First sample)
A triangular pyramid created using graph paper was used as a sample. Graph paper memory is 1 mm apart. The photo was taken while focusing on the apex of the triangular pyramid.

(Second sample)
Porous silica beads used as a desiccant were used as samples. The bead diameter is 3 mm to 7 mm. Samples are mixed with beads of different sizes.

<Results and discussion>
The observation results of the first sample (triangular pyramid made of graph paper) are shown in FIG. As shown in FIG. 12, it was clearly confirmed that the larger the aperture diameter adjusted by the diaphragm, the shallower the focal depth, and the smaller the aperture diameter, the deeper the focal depth.

The observation result of the second sample (porous silica beads) is shown in FIG. Image acquisition was performed by focusing on the position indicated by the square in FIG. Similar to the case of FIG. 12, also in the case of FIG. 13, it was clearly confirmed that the larger the aperture diameter adjusted by the diaphragm, the shallower the focal depth, and the smaller the aperture diameter, the deeper the focal depth.

Thus, the effect of the diaphragm was shown, and it became clear that the image reflecting the depth information was obtained as the aperture diameter of the diaphragm was smaller.

[Second Embodiment]
<Observation method>
A mouse femur sample was observed using an optical system having XLFLUOR4X / 340 similar to that of the first example. In this example, a two-dimensional image was obtained by photographing in a state where no diaphragm was attached and in a state where a diaphragm with an aperture diameter of 8.4 mm was attached.

As a sample of the mouse femur, the sample used in Okuboe et al., “PLoS” ONE 8 (11) 2013, “DOI: 10.1371 / journal.pone.0078306” was used. That is, the femur of the clock gene Per2 :: luciferase knock-in mouse was used. The femur was cultured in a 35 mm dish. Observation was performed by adding luciferin to the dish.

In this example, a bright field image and a light emission image were acquired. When acquiring a bright field image, the exposure time was set to 50 milliseconds. When acquiring the luminescent image, the exposure time was 3 minutes or 10 minutes.

<Results and discussion>
The observation results of this example are shown in FIG. As shown in the luminescence image without the diaphragm, without the diaphragm, the luminescence of the femoral head, which is the tip of the mouse femur, with a high luminescence amount indicated by the arrow was saturated, and the femoral body could not be observed. . On the other hand, when the aperture diameter was φ8.4 mm, the entire femoral head and femoral body sample luminescence including the portion indicated by the square in the image with an exposure time of 10 minutes could be acquired. Also in the luminescent image, it was clearly confirmed that the larger the aperture diameter adjusted by the diaphragm, the shallower the focal depth, and the smaller the aperture diameter, the deeper the focal depth.

From this, it was found that the optical aperture value can be selectively determined according to the target sample so that the light emission is not saturated and the depth information of the sample can be acquired as much as possible. In this way, continuous three-dimensional analysis, which has been difficult in the past, can be easily realized by repeatedly acquiring two-dimensional luminescent images regarding the thick femoral head and the entire femoral body sample. If the sample has a brightness value that changes within the allowable upper and lower limits, regardless of the rate of change in the brightness value of the light emission, the weak light is repeatedly emitted using the same optical aperture selected. It is considered that a three-dimensional time series analysis can be performed by acquiring an image obtained by the above method. In this case, a three-dimensional image composed of a plurality of two-dimensional images is formed using an aperture diameter relatively larger than the selected optical aperture, and depth information is acquired from the three-dimensional image to obtain a three-dimensional image. It is possible to provide an analysis method and an analysis system capable of executing analysis continuously and with high accuracy.

Claims (11)

1. An image acquisition process for acquiring a weak light image of a weakly luminescent sample as a three-dimensional sample prepared to emit weak light and including a plurality of analysis objects;
   Analyzing the three-dimensional sample based on the acquired weak light image,
   The image acquisition process includes:
   Determining an aperture diameter of an optical diaphragm inserted in an optical system for acquiring the weak light image based on a predetermined condition;
   Repeatedly performing a weak light image on the three-dimensional sample using the optical aperture of the determined aperture diameter,
   In at least one of the image acquisition processes that are repeatedly performed, the aperture diameter is a value that includes the analysis target existing in a different position in the optical axis direction of the optical system within the depth of focus. Determined to
   Method for analyzing weakly luminescent samples.
2. The initial value of the opening diameter is the first opening diameter;
   The condition is whether or not the change in luminance value of the weak light image is greater than a predetermined value,
   The determination of the aperture diameter is performed when the weak light image repeatedly acquired with the first aperture diameter is compared, and the change in the luminance value of the weak light image is larger than the predetermined value. Determining a second opening diameter that is larger than the first opening diameter;
   Acquiring the weak light image includes acquiring a set of two-dimensional weak light images for a plurality of different focusing surfaces when the opening diameter is the second opening diameter,
   The image acquisition process further includes synthesizing the set of two-dimensional weak light images to generate a three-dimensional weak light image.
   The method for analyzing the weakly luminescent sample according to claim 1.
3. The condition is whether or not the elapsed time has become a preset timing,
   Determining the opening diameter is
   Determining the opening diameter to be a first opening diameter at a repeated first timing;
   Determining the opening diameter to be a second opening diameter larger than the first opening diameter at a repeated second timing;
   Including
   Acquiring the weak light image includes acquiring a set of two-dimensional weak light images for a plurality of different focusing surfaces when the opening diameter is the second opening diameter,
   The image acquisition process further includes synthesizing the set of two-dimensional weak light images to generate a three-dimensional weak light image.
   The method for analyzing the weakly luminescent sample according to claim 1.
4. The analysis process is
   Displaying a plurality of the weak light images obtained with the first opening diameter on a display;
   Using the three-dimensional weak light image synthesized based on a set of two-dimensional weak light images acquired at a timing closest to the weak light image selected from the plurality of displayed weak light images The method for analyzing a weakly luminescent sample according to claim 3, comprising performing a predetermined analysis.
5. The initial value of the opening diameter is the first opening diameter;
   The image acquisition process further includes displaying an image based on the weak light image acquired at the first aperture diameter on a display;
   Determining the opening diameter includes determining the opening diameter to be the second opening diameter when an instruction to make the second opening diameter larger than the first opening diameter is input.
   The method for analyzing the weakly luminescent sample according to claim 1.
6. Acquiring the weak light image includes acquiring a set of two-dimensional weak light images for a plurality of different focusing surfaces when the opening diameter is the second opening diameter,
   The image acquisition process further includes synthesizing the set of two-dimensional weak light images to generate a three-dimensional weak light image.
   The method for analyzing a weakly luminescent sample according to claim 5.
7. The method for analyzing a weakly luminescent sample according to claim 1, wherein the three-dimensional sample includes a plurality of cells.
8. An objective optical system;
   An optical aperture provided in the objective optical system;
   An aperture determining unit that determines an aperture diameter of the optical diaphragm based on a predetermined condition;
   An aperture drive unit for setting the aperture diameter of the optical aperture to a determined value;
   A weak light image of a weakly luminescent sample as a three-dimensional sample including a plurality of analysis objects prepared so as to emit weak light in a state where the optical aperture is the determined aperture diameter, and the objective optical An analysis system comprising: an imaging device that images through the system.
9. A drive unit for moving the in-focus position of the objective optical system along the optical axis;
   A shooting control unit that controls the operation of the imaging device while controlling the operation of the driving unit;
   An image composition unit, and
   The condition is whether or not the change in luminance value of the weak light image is greater than a predetermined value,
   The opening determination unit sets the initial value of the opening diameter as a first opening diameter, and the brightness value of the weak light image based on a comparison result of the weak light image repeatedly acquired with the first opening diameter. When the change of becomes larger than the predetermined value, the second opening diameter larger than the first opening diameter is determined,
   When the aperture diameter is the second aperture diameter, the imaging control unit causes the imaging device to acquire a set of two-dimensional weak light images for a plurality of different focusing surfaces,
   The image synthesis unit synthesizes the set of two-dimensional weak light images to generate a three-dimensional weak light image;
   The analysis system according to claim 8.
10. A drive unit for moving the in-focus position of the objective optical system along the optical axis;
    A shooting control unit that controls the operation of the imaging device while controlling the operation of the driving unit;
    An image composition unit, and
    The condition is whether or not the elapsed time has become a preset timing,
    The opening determining unit
    The opening diameter is determined to be the first opening diameter at the first timing repeated,
    The opening diameter is determined to be a second opening diameter larger than the first opening diameter at a second timing repeated;
    When the aperture diameter is the second aperture diameter, the imaging control unit causes the imaging device to acquire a set of two-dimensional weak light images for a plurality of different focusing surfaces,
    The image synthesis unit synthesizes the set of two-dimensional weak light images to generate a three-dimensional weak light image;
    The analysis system according to claim 8.
11. A display device for displaying an image;
    A display control unit for controlling display on the display device;
    An image analysis unit that performs analysis based on the image,
    The display control unit causes the display device to display a plurality of the weak light images obtained with the first opening diameter,
    The image analysis unit is configured to combine the three-dimensional image based on a set of two-dimensional weak light images acquired at a timing closest to the weak light image selected from the displayed weak light images. Perform predetermined analysis using weak light images,
    The analysis system according to claim 10.

Images