Optical signal detection device, gel member, and optical signal detection method

Abstract

To allow an excellent observation by using a liquid immersion objective lens while reducing the load on a user.SOLUTION: A microscope device 1 includes: a liquid immersion objective lens 3; a container C as a holding member for holding a sample S between the objective lens 3 and a sample S; and a gel member 10 for filling a space between the objective lens 3 and the container C. The gel member 10 has a 1 / 4 consistency showing a value in the range of 44 to 111, both inclusive, measured by a consistency test using a JIS K 2220 1 / 4 circular cone.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The disclosure of the present specification relates to an optical signal detection device, a gel member, and an optical signal detection method. [Background technology]

[0002] Currently, research using cell aggregates such as spheroids and organoids, which are made by culturing a large number of cells in three dimensions, is attracting attention. These samples are typically between 100 and 500 μm in size.

[0003] For deep cell observation of such samples, immersion objectives are generally used, which can achieve a higher numerical aperture than dry objectives by filling the space between the objective and the sample (or more precisely, the holding member that holds the sample) with immersion liquid.

[0004] By using an immersion liquid with a refractive index close to that of the sample, it is also possible to suppress spherical aberration caused by refractive index mismatch that occurs at the interface between the sample and the immersion liquid (or air in the case of a dry objective lens). As described in Patent Document 1 and elsewhere, the effect of spherical aberration caused by refractive index mismatch becomes more pronounced the deeper the observation position, so suppressing spherical aberration makes it possible to observe at deeper positions. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2017-026666 Summary of the Invention [Problem to be solved by the invention]

[0006] In actual observations, dry and immersion objectives are sometimes switched during the course of an observation. For example, a typical example is to first locate a region of interest using a dry objective with a relatively low magnification and wide field of view, and then observe that region in detail using an immersion objective with high resolution.

[0007] However, when switching from a dry objective to an immersion objective, it is necessary to supply new immersion liquid and fill the space between the objective and the sample with it. Furthermore, when switching from an immersion objective to a dry objective, the immersion liquid must be carefully wiped off to ensure that no residue remains on the sample surface. Thus, switching between dry and immersion objectives requires various additional tasks in addition to the task of switching the objectives themselves. This temporarily interrupts observation, making smooth observation difficult.

[0008] Observing samples using only immersion objectives also presents various challenges compared to using dry objectives. For example, if water is used as the immersion liquid, the immersion liquid will evaporate over long periods of observation. Therefore, immersion liquid must be supplied as needed during observation. Furthermore, if oil is used as the immersion liquid, cleaning is time-consuming and bubbles are likely to form due to its high viscosity. Furthermore, when using an objective with a long working distance in an inverted microscope, maintaining the immersion liquid between the objective and the sample using surface tension is difficult. Maintaining the immersion liquid using surface tension is also difficult when observing samples from oblique or lateral angles, presenting similar challenges to those encountered with long-working-distance objectives. Therefore, in these cases, a complex mechanism is required to maintain the immersion liquid between the objective and the sample. Furthermore, when observing a wide depth range, the distance between the objective and the sample changes significantly during observation, which can lead to the immersion liquid spilling out from between the objective and the sample.

[0009] As described above, the current situation is that the use of an immersion objective lens imposes a greater burden on the user than the use of a dry objective lens. In light of the above situation, an object of one aspect of the present invention is to provide a technology that enables good observation using an immersion objective lens while reducing the burden on the user. [Means for solving the problem]

[0010] An optical signal detection device according to one embodiment of the present invention includes an objective lens, a holding member that holds the sample between the objective lens and the sample, and a light source that fills a space between the objective lens and the holding member. Silicone gel and Silicone gel has a 1 / 4 consistency of 44 or more and 111 or less, measured based on the consistency test using a 1 / 4 cone of JIS K 2220.

[0012] In an optical signal detection method according to one embodiment of the present invention, a space between an objective lens and a holding member for holding a sample is filled with a 1 / 4 cone having a 1 / 4 consistency of 44 or more and 111 or less, as measured based on a consistency test using a 1 / 4 cone of JIS K 2220. Silicone gel and detecting light from the sample that has passed through the objective lens and is incident on the sample with a photodetector. [Effects of the Invention]

[0013] According to the above aspect, it is possible to provide a technique that enables good observation using an immersion objective lens while reducing the burden on the user. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram illustrating a configuration of a microscope apparatus according to an embodiment. [Figure 2] FIG. 1 is a diagram for explaining a method for measuring consistency. [Figure 3] FIG. 4 is a diagram illustrating the shape of a gel member. [Figure 4]10A and 10B are diagrams showing examples of the shape of a gel member. [Figure 5] 10A and 10B are diagrams showing another example of the shape of the gel member. [Figure 6] FIG. 10 is a diagram showing yet another example of the shape of the gel member. [Figure 7] 10A and 10B are diagrams for explaining a method for attaching and detaching a gel member to and from an objective lens. [Figure 8] FIG. 2 is a diagram showing an example of the relationship between a sample and a medium in a container. [Figure 9] FIG. 10 is a diagram showing another example of the relationship between a sample and a medium in a container. [Figure 10] 10 is a table showing the results of a comparison between a case where oil is used as an immersion liquid and a case where a gel member is used. [Figure 11] 10 is another table showing the results of a comparison between a case where oil is used as the immersion liquid and a case where a gel member is used. [Figure 12] FIG. 1 is a diagram illustrating the configuration of a microscope device used in an experiment. [Figure 13] FIG. 10 is a diagram illustrating the configuration of another microscope device used in the experiment. [Figure 14] This is a microscope image taken in Experiment 1 using oil as the immersion liquid. [Figure 15] This is a microscope image taken in Experiment 1 using a gel member as the immersion liquid. [Figure 16] This is a microscope image taken in Experiment 10 using a 4x dry objective. [Figure 17] This is a microscope image taken in experiment 10 using a 10x dry objective. [Figure 18] This is a microscope image taken in experiment 10 using a 20x dry objective. [Figure 19] This is a microscope image taken in Experiment 10 using a 30x immersion objective lens. [Figure 20] FIG. 10 is a diagram showing an example of observation from an oblique direction. [Figure 21] FIG. 1 is a perspective view of a microplate. [Figure 22]FIG. 2 is a diagram illustrating the structure of a microplate. DETAILED DESCRIPTION OF THE INVENTION

[0015] FIG. 1 illustrates the configuration of a microscope apparatus according to one embodiment. The microscope apparatus 1 shown in FIG. 1 is an inverted microscope for observing a sample S from below and is an example of an optical signal detection apparatus for detecting optical signals. As shown in FIG. 1, the microscope apparatus 1 includes a light source 2, an objective lens 3 attached to a revolver 4, and a camera 5. The objective lens 3 is a so-called immersion objective lens that is used with the space between the objective lens 3 and a container filled with immersion liquid. A container C that contains the sample S is, for example, a glass-bottom dish, and is an example of a holding member that holds the sample S. The sample S is not particularly limited, but may be, for example, a biological sample such as cells. The sample S may also be a sample having a thickness of several hundred micrometers, such as a three-dimensionally cultured spheroid or organoid.

[0016] 1 shows an example in which the microscope device 1 is an inverted microscope, the microscope device 1 may also be an upright microscope. In this case, the gel member 10 may fill the space between the objective lens 3 and a cover glass, which is another example of a sample holding member.

[0017] The microscope device 1 acquires an image of the sample S by irradiating the sample S with light from a light source 2 and detecting the light from the sample S with a camera 5. The observation method of the microscope device 1 is not particularly limited. For example, the image of the sample S may be acquired by bright-field observation or fluorescence observation. Other observation methods may also be used, such as phase contrast observation or differential interference observation.

[0018] The microscope device 1 includes a gel member 10 between the objective lens 3 of the immersion system and the container C, which has a higher refractive index than air, instead of a liquid immersion liquid.

[0019] Unlike immersion liquid, the gel member 10 does not have fluidity. Therefore, compared to immersion liquid, which is held between the objective lens 3 and the container C by surface tension, the gel member 10 can be easily placed in the observation optical path between the objective lens 3 and the container C, and it is also easy to remove from the observation optical path. Therefore, even when switching between an immersion objective lens and a dry objective lens, the switching process can be performed quickly. Furthermore, unlike immersion liquid, the gel member 10 does not flow off the objective lens 3 during observation. Therefore, it does not contaminate the objective lens 3 or its surroundings, and therefore cleaning after use is easy. Furthermore, since there is no significant volume loss due to evaporation, there is no need to supply additional gel during observation as with immersion liquid, and it can easily be used for long-term observation. Furthermore, because the gel member 10 has relatively high adhesiveness, it can remain stably between the objective lens 3 and the container C even when observed from an oblique or sideways angle. As such, the gel member 10 is much easier to handle than immersion liquid, significantly reducing the burden on users when using immersion objective lenses.

[0020] Furthermore, like the immersion liquid, the gel member 10 has a refractive index higher than that of air. This allows the immersion objective lens 3 to perform at its full potential, achieving a high numerical aperture and enabling a bright image with high resolution to be obtained. The difference in refractive index between the gel member 10 and the container C or the sample S is also smaller than when air is present. Therefore, like the case where immersion liquid is used, it is possible to observe the sample S to its depths while suppressing spherical aberration caused by the difference in refractive index between the container C or the sample S. Therefore, by using the gel member 10, the three-dimensional structure of the sample can be observed well.

[0021] As described above, the microscope apparatus 1, which performs observation using the gel member 10 instead of immersion liquid, allows for good observation of the sample S without imposing an excessive burden on the user, even when using the immersion objective lens 3. Furthermore, since switching between the dry objective lens and the immersion objective lens can be performed smoothly, interruptions to observation due to objective lens switching can be kept to a short time, allowing for efficient observation.

[0022] FIG. 2 is a diagram for explaining a consistency measurement method. FIG. 3 is a diagram for explaining the shape of the gel member. FIGS. 4 to 6 are diagrams showing examples of the shape of the gel member. FIG. 7 is a diagram for explaining a method for attaching and detaching the gel member to and from the objective lens. A desirable configuration of the microscope device 1 will be described below with reference to FIGS. 2 to 7.

[0023] The gel member 10 included in the microscope device 1 desirably has a 1 / 4 penetration value of 44 or more and 111 or less, measured based on the penetration test using a 1 / 4 cone of JIS K 2220 described above. Generally, as defined in JIS K 2220, penetration refers to the distance a standard cone and an optional cone penetrate into a sample under specified conditions of load, time, and temperature, and is expressed as a value measured in 0.1 mm increments multiplied by 10. Meanwhile, 1 / 4 penetration refers to the 1 / 4 scale penetration defined in JIS K 2220, and is the penetration measured using a specified cone (1 / 4 cone) that is a quarter-scale reduction of the standard cone or optional cone. That is, as shown in FIG. 2, penetration refers to the distance L that a 1 / 4 cone 6 penetrates into a test object 7 under specified conditions of load, time, and temperature, and is expressed as a value measured in 0.1 mm increments multiplied by 10. The measurements are carried out at a temperature of 25°C ± 0.5°C.

[0024] If the 1 / 4 consistency of the gel member 10 exceeds the upper limit of 111, the gel member 10 becomes too soft for use as a substitute for immersion liquid. This makes it difficult for the gel member 10 to maintain its shape and causes it to collapse. As a result, the space between the objective lens 3 and the container C cannot be filled with the gel member 10, which can lead to poor observation. This problem is particularly likely to occur when observing the sample S using an objective lens 3 with a long working distance (e.g., WD = 4 mm) and with a long distance between the objective lens 3 and the container C. Furthermore, if the gel member 10 is too soft, when a gel member 10 of a predetermined shape is created using a mold, the gel member 10 will not maintain its shape when released from the mold, resulting in an increased rate of defective products. Furthermore, its durability is reduced, limiting the number of times it can be reused.

[0025] On the other hand, if the 1 / 4 consistency of the gel member 10 is below the lower limit of 44, the gel member 10 will be too hard for use as a substitute for immersion liquid. If the gel member 10 has an appropriate hardness, it will deform in accordance with the distance between the objective lens 3 and the container C, allowing the gel member 10 to appropriately fill the space between the objective lens 3 and the container C without applying excessive pressure to the container C. In contrast, if the gel member 10 is too hard, i.e., if the 1 / 4 consistency of the gel member 10 is below the lower limit of 44, the gel member 10 will not flexibly deform even when the distance between the objective lens 3 and the container C changes. For this reason, for example, when the objective lens 3 is brought closer to the container C for deep observation, the gel member 10 pressed by the objective lens 3 will not sufficiently expand between the objective lens 3 and the container C, and the distance between the objective lens 3 and the container C will not decrease to the expected distance. This can cause problems such as a large force being applied to the container C, causing the container to deform or shift in position, etc. As a result, the observation position also shifts, making it difficult to correctly observe the region of interest.

[0026] By setting the 1 / 4 consistency of the gel member 10 to a value of 44 or more and 111 or less, the microscope apparatus 1 can perform appropriate observation without causing any inconvenience even when the distance between the objective lens 3 and the container C changes. Furthermore, even when the positional relationship between the objective lens 3 and the container C changes in a direction perpendicular to the optical axis, the microscope apparatus 1 can perform appropriate observation without causing any inconvenience. Therefore, the microscope apparatus 1 can maintain the above-mentioned effect of observing the sample S well over a wide observation range without imposing an excessive burden on the user.

[0027] In the gel member 10 that fills the space between the objective lens 3 and the container C, it is desirable that the contact area of ​​the gel member 10 with the objective lens 3 (hereinafter referred to as the first contact area) is larger than the contact area of ​​the gel member 10 with the container C (hereinafter referred to as the second contact area), as shown in Fig. 3. For this reason, the gel member 10 may have, for example, a truncated cone shape as shown in Fig. 4, and in that case, it is desirable to place the gel member 10 with the apex of the cone facing the objective lens 3. Note that Fig. 3 shows the gel member 10 in contact with the container C in a circular area with a radius R1, and in contact with the objective lens 3 in a circular area with a radius R2 (>radius R1).

[0028] Because the first contact area is larger than the second contact area, the gel member 10 can adhere more firmly to the objective lens 3 than to the container C. This allows the gel member 10 to peel off from the container C and remain on the objective lens 3 when the distance between the objective lens 3 and the container C is increased to a value equal to or greater than the thickness of the gel member 10. Therefore, according to the microscope device 1, when the objective lens 3 is switched to another objective lens, the gel member 10 adsorbed to the objective lens 3 moves out of the optical path together with the objective lens 3 as the revolver rotates, which eliminates the need for the user to remove the gel member 10 and allows for smooth switching of the objective lens.

[0029] 4 illustrates a gel member 10 having a truncated cone shape, but the shape of the gel member of the microscope device 1 is not limited to a truncated cone shape like the gel member 10. For example, the microscope device 1 may include a cylindrical gel member 11 as shown in FIG. 5 instead of the gel member 10, or may include a gel member having a prismatic or truncated pyramidal shape.

[0030] 6, the microscope device 1 may also include a gel member 12 that has a bottom surface and a convex surface formed by a curved surface, in which case it is desirable that the gel member 12 have the convex surface facing the container C. As shown in gel members 10 and 12, the gel member has a tapered shape in which the cross-sectional area decreases from the objective lens 3 toward the container C, which makes it easy to achieve the relationship in size between the contact areas described above. Therefore, from the perspective of simplifying the objective lens switching operation, it is desirable that the gel member has a tapered shape in which the cross-sectional area decreases from the objective lens 3 toward the container C.

[0031] As described above, the gel member has adhesiveness and is removably attached to the objective lens. Therefore, when attaching the gel member to the objective lens 3, it is sufficient to simply place the gel member (here, gel member 12) on the tip 3a of the objective lens 3 using tweezers or the like, as shown in FIG. 7. This causes the gel member 12 to be attached to the objective lens 3, making it easy to attach the gel member 12 to the objective lens 3. Similarly, tweezers or the like can be used to detach the gel member 12 from the objective lens 3. In this way, because the gel member 12 is removably attached to the objective lens 3, preparations for observation can be easily performed.

[0032] A more desirable configuration of the microscope apparatus 1 will be described below. The thickness of the gel member (e.g., gel member 10) of the microscope apparatus 1 is preferably 1.1 to 1.5 times the working distance of the objective lens 3. When the thickness of the gel member 10 is 1.1 times the working distance or more, the space between the container C and the objective lens 3 can be completely filled with the gel member 10, even taking into account the thickness of the container C, and the surface of the sample S (the bottom surface of the sample S in FIG. 1 ) can be observed. Furthermore, when the thickness of the gel member 10 is 1.5 times the working distance or less, excessive compression of the gel member 10 can be avoided when observing a deep portion of the sample S. If the gel member 10 is compressed too much, a large pressure will be applied to the container C, causing the container C to deform or the position of the container C to shift. As a result, the observation position will shift, making it difficult to accurately observe the region of interest. When the thickness is 1.5 times the working distance or less, the gel member 10 will not be subjected to a large pressure, reducing deformation of the container C or shifting of the position of the container C, and allowing observation at the intended position. Therefore, even in Z-stack imaging, which is performed by scanning the sample S in the depth direction, an image can be acquired at the intended position while the space between the container C and the objective lens 3 is filled with the gel member 10 without any gaps.

[0033] Furthermore, it is desirable that the refractive index difference between the gel member 10 and the container C be within ±0.1. By keeping the refractive index difference within ±0.1, spherical aberration caused by refractive index mismatch can be suppressed, allowing for good observation. This effect is particularly evident in deep observation, making it particularly suitable for observing thick samples. The standard refractive index of the bottom surface of a glass-bottom dish or cover glass is 1.52. Therefore, if there is a large variation in the thickness of the glass-bottom dish or cover glass, the gel member 10 may have a refractive index in the range of 1.52 ±0.1, for example. This allows for the reduction of spherical aberration caused by thickness variations without using a correction collar mechanism for the objective lens.

[0034] Furthermore, it is desirable that the refractive index difference between the gel member 10 and the sample S or the medium M covering the sample S be within ±0.1. FIGS. 8 and 9 are diagrams illustrating the relationship between the sample and the medium in a container. The medium M is not particularly limited, but may be, for example, a culture medium or a clearing solution. By keeping the refractive index difference within ±0.1, spherical aberration caused by refractive index mismatch that occurs during deep observation can be suppressed, allowing for good observation. As shown in FIGS. 8 and 9, the sample S may be in contact with the bottom surface of the container C or may be separated from the bottom surface of the container C. Even when the sample S is separated from the bottom surface of the container C via the medium M, as long as the refractive index difference is within ±0.1, spherical aberration caused by refractive index mismatch that occurs during observation of the sample S through the medium M can be suppressed, allowing for good observation of the sample S.

[0035] 10 and 11 are tables showing the results of a comparison between the use of oil and the use of a gel member as the immersion liquid. Below, with reference to Fig. 10 and Fig. 11, the results of a comparison between the use of a gel member having a 1 / 4 consistency in the range of 44 to 111, which the inventors of the present application have found to be a desirable range, and the use of oil will be described.

[0036] Table T1 in Figure 10 and Table T2 in Figure 11 show the relationship between the reference z coordinate (Z) based on the bottom surface of sample S when observing using oil and the observation z coordinate (P) indicating the Z coordinate of the microscope device at that time, and the relationship between the reference z coordinate (Z) and the observation z coordinate (P) when observing using a gel member. The unit of the z coordinate is μm. Note that the combination of Z and P when using a gel member is the combination of Z and P when an image is acquired that is sufficiently similar to the image acquired with the combination of Z and P when using oil. Furthermore, the results in Figures 10 and 11 were obtained using the microscope in Figure 12, which will be described later.

[0037] Five types of gel materials were used: 55, 62, 69, 91, and 111 in consistency. The consistency values ​​were all 1 / 4 consistency. Table T1 shows that z-stack imaging was performed six times with a gel material with a consistency of 55 (gel number 1). It also shows that z-stack imaging was performed six times, four times, and five times with a gel material with a consistency of 62 (gel number 2), a gel material with a consistency of 69 (gel number 3), and a gel material with a consistency of 91 (gel number 4). Table T2 also shows that four gel materials with a consistency of 111 were prepared (gel numbers 5 to 8), and z-stack imaging was performed twice for each.

[0038] First, let's look at the results for the gel element with a consistency of 55 (gel number 1) in Table T1. The results of the first z-stack imaging were obtained when the gel element had not yet been crushed, and therefore the conditions are significantly different from those of the second and subsequent z-stack imaging, so they will be excluded from consideration. We will now consider the results of the second and subsequent imaging. Note that in the first z-stack imaging, the positions measured were the same as the images obtained when oil was used at depths of 0 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, and 300 μm.

[0039] For the gel element with a consistency of 55 (gel number 1), the maximum variation in the z-axis position where the image was taken from the second or subsequent run was 7 μm. In contrast, for the gel element with a consistency of 62 (gel number 2), the gel element with a consistency of 69 (gel number 3), the gel element with a consistency of 91 (gel number 4), and the gel elements with a consistency of 111 (gel numbers 5-8), the maximum variations in the z-axis position were 5 μm, 3 μm, 2 μm, and 1 μm, respectively.

[0040] These results indicate that the lower the consistency, i.e., the harder the gel component, the greater the variation in the observation position. This is presumably because the harder the gel component, the greater the pressure applied to the container and sample when the gel component deforms to a shape corresponding to the distance between the objective lens and the container, resulting in changes in the position of the container and sample. On the other hand, assuming a cell size of 20 μm, if the variation in the observation position can be reduced to half that, 10 μm, it will be possible to capture and observe the same cell in repeated observations. Therefore, from the measurement results shown in Figures 10 and 11, it can be determined that the variation can be reduced to 10 μm if the consistency is 44 or higher, the lower limit of the desired 1 / 4 consistency range described above. In other words, by using a gel component that satisfies the desired 1 / 4 consistency range described above, reproducibility of the observation position can be ensured.

[0041] Next, let's look at the results in Table T2. These are the results of Z-stack imaging performed twice on four gel components with the same consistency of 111. Although not shown in Table T2, the Z position was stable, and with the exception of gel number 7, the third Z-stack imaging yielded the same Z position as the second Z-stack imaging for each gel. However, gel number 7 could not be imaged a third time due to its shallow portion. This was because the gel was crushed during the second deep region observation, and the gel shape did not return to normal during the third shallow region observation of the sample, making it impossible to fill the space between the container and the objective lens with gel components.

[0042] From the above results, it can be seen that with a gel having a consistency of 111, the gel may not be restored after being crushed, making it impossible to repeatedly observe shallow regions of the sample, but since 75% of the observations could be repeated, it can be said that the gel member can be used repeatedly if the consistency of 111 is set as the upper limit. Therefore, if the gel member satisfies the above-mentioned desirable 1 / 4 consistency range, the gel member can change its shape depending on the distance between the container and the objective lens, even when used repeatedly, and can fill the space between the container and the objective lens.

[0043] As described above, it is desirable that the gel member has a 1 / 4 consistency that is equal to or greater than 44 and equal to or less than 111.

[0044] Figures 12 and 13 are diagrams explaining the configuration of the microscope apparatus used in the experiments. Figure 14 is a microscope image acquired in Experiment 1 using oil as the immersion liquid, and Figure 15 is a microscope image acquired in Experiment 1 using a gel member as the immersion liquid. Figures 16 to 18 are microscope images acquired in Experiment 10 using 4x, 10x, and 20x dry objective lenses, respectively. Figure 19 is a microscope image acquired in Experiment 10 using a 30x immersion objective lens.

[0045] Various experiments conducted by the inventors will be described below with reference to Figures 12 to 19, and the results will be discussed. First, the microscope device used in the experiments will be described with reference to Figures 12 and 13.

[0046] The microscope 100 shown in Fig. 12 is an inverted microscope and an example of an optical signal detection device that detects an optical signal. The microscope 100 has a halogen lamp 101 as a light source, and is provided, on an illumination optical path from the halogen lamp 101 to a sample S, with a bandpass filter 102 that transmits light in the 1100±25 nm wavelength range, a diffuser 103, a collimator lens 104, a window lens 105, and a collector lens 106. The microscope 100 also has a camera 109 with a CMOS (Complementary Metal Oxide Semiconductor) image sensor with enhanced sensitivity in the near-infrared wavelength range, and is provided, on an observation optical path from the sample S held in a container C to the camera 109, with an objective lens 107 and an imaging lens 108.

[0047] The objective lens 107 is an immersion type objective lens. The space between the objective lens 107 and the container C is filled with the gel member 10 described above.

[0048] The microscope apparatus 100 acquires an image by bright-field observation. Specifically, the microscope apparatus 100 irradiates the sample S with near-infrared light that is less likely to scatter and that has passed through the band-pass filter 102, with the space between the objective lens 107 and the container C filled with a gel member 10 having a ¼ consistency value of 44 or more and 111 or less, and detects the light from the sample S that has entered via the objective lens 107 with the camera 109, thereby acquiring an image of the sample S. Furthermore, the microscope apparatus 100 may change the distance in the optical axis direction between the objective lens 107 and the sample S (container C), and repeat the irradiation of the sample S with light and the detection of the light from the sample S each time the distance is changed.

[0049] The microscope 200 shown in FIG. 13 is an inverted microscope, more specifically, a two-photon excitation laser scanning microscope, which is an example of an optical signal detection device that detects optical signals. The microscope 200 includes a laser 201 as a light source, and includes a scanner 202, a relay lens 203, a mirror 204, a dichroic mirror 205, and an objective lens 206 on an illumination optical path from the laser 201 to a sample S. The scanner 202 is a two-dimensional scanner that deflects illumination light in two directions perpendicular to the optical axis of the objective lens 206, and may include, for example, a galvanometer scanner and a resonant scanner. The microscope 200 also includes a photodetector 208, and includes an objective lens 206, a dichroic mirror 205, and a relay lens 207 that relays the pupil of the objective lens 206 to the photodetector 208 on an observation optical path from the sample S held in a container C to the photodetector 208.

[0050] The objective lens 206 is an immersion type objective lens. The space between the objective lens 206 and the container C is filled with the gel member 10 described above.

[0051] The microscope device 200 acquires an image by two-photon excitation fluorescence observation. Specifically, the microscope device 200 irradiates the sample S with laser light having an excitation wavelength of 700 nm while moving the irradiation position two-dimensionally with the scanner 202 in a state where the space between the objective lens 206 and the container C is filled with a gel member 10 having a ¼ consistency value of 44 or more and 111 or less, and acquires an image of the sample S based on the signal detected by the photodetector 208 and information on the irradiation position by detecting the fluorescence from the sample S that has entered via the objective lens 206. Furthermore, the microscope device 200 may change the distance in the optical axis direction between the objective lens 206 and the sample S (container C), and repeat the irradiation of the sample S with light and the detection of the fluorescence from the sample S each time the distance is changed.

[0052] A total of 11 experiments were conducted, Experiments 1 to 11, and it was confirmed that good observations could be made using the gel material in all cases. Whether good observations were made or not was confirmed for Experiments 1-4 and 7-8 by comparing images taken using the gel material with images taken using an immersion liquid such as water or oil. For Experiments 5-6 and 9-11, confirmation was made using the images taken using the gel material alone. Details of each experiment are as follows.

[0053] <Experiment 1> Observation pattern: Photographed at reference z coordinates Z = 52 μm, 156 μm, and 263 μm (photographed at z coordinate positions where images similar to those photographed at each reference z coordinate position of the comparison immersion liquid are obtained) Immersion liquid and position for comparison: Silicone oil Reference z coordinate Z = 50 μm, 150 μm, 250 μm Objective lens: 30x, NA1.05, WD0.8mm oil immersion objective lens Gel material: Silicone gel Gel material consistency (1 / 4 consistency): 69 Refractive index of gel material: 1.405 Shape of gel component: rectangular parallelepiped, 4mm long x 4mm wide x 0.9mm thick Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 1100nm Container: 35mm glass bottom dish Weight: Approx. 210g Sample: HT29 cell spheroid, approximately 350 μm thick

[0054] The microscope image P1 shown in Fig. 14 is a bright-field image of the sample acquired using silicone oil at Z = 150 μm. The microscope image P2 shown in Fig. 15 is a bright-field image of the sample acquired using a gel member at Z = 156 μm. From Figs. 14 and 15, it can be seen that even when using a gel member, images comparable to those obtained when using silicone oil can be obtained.

[0055] <Experiment 2> Observation pattern: Photographed at reference z coordinates Z = 51 μm, 152 μm, and 255 μm (photographed at z coordinate positions where images similar to those photographed at each reference z coordinate position of the comparison immersion liquid are obtained) Immersion liquid and position for comparison: Silicone oil Reference z coordinate Z = 50 μm, 150 μm, 250 μm Objective lens: 30x, NA1.05, WD0.8mm oil immersion objective lens Gel material: Silicone gel Gel material consistency (1 / 4 consistency): 111 Refractive index of gel material: 1.405 Shape of gel component: rectangular parallelepiped, 4mm long x 4mm wide x 0.9mm thick Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 1100nm Container: 35mm glass bottom dish Weight: Approx. 210g Sample: HT29 cell spheroid, approximately 350 μm thick

[0056] <Experiment 3> Observation pattern: Photographed at reference z coordinates Z = 52 μm, 156 μm, and 260 μm (photographed at z coordinate positions where images similar to those photographed at each reference z coordinate position of the comparison immersion liquid are obtained) Immersion liquid and position for comparison: Silicone oil Reference z coordinate Z = 50 μm, 150 μm, 250 μm Objective lens: 30x, NA1.05, WD0.8mm oil immersion objective lens Gel material: Silicone gel Gel material consistency (1 / 4 consistency): 69 Refractive index of gel material: 1.405 Shape of gel component: Φ6 x maximum thickness 1.1 mm (curvature 4.64 mm) spherical indentation Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 1100nm Container: 35mm glass bottom dish Weight: Approx. 210g Sample: HT29 cell spheroid, approximately 350 μm thick

[0057] <Experiment 4> Observation pattern: Photographed at reference z coordinates Z = 49 μm, 148 μm, and 246 μm (photographed at z coordinate positions where images similar to those photographed at each reference z coordinate position of the comparison immersion liquid are obtained) Immersion liquid and position for comparison: Water Reference z coordinate Z = 49 μm, 149 μm, 245 μm Objective lens: 25x, NA1.05, WD2mm water immersion objective lens Gel material: Aqua Joint Gel material consistency (1 / 4 consistency): 96 Refractive index of gel material: 1.356 Shape of gel component: truncated cone with top diameter 3mm, bottom diameter 7mm, and thickness 2.5mm Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 1100nm Container: 35mm glass bottom dish Weight: Approx. 130g Sample: HT29 cell spheroid, approximately 350 μm thick

[0058] <Experiment 5> Observation pattern: Photographed at reference z coordinate Z = 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm Immersion fluid for comparison: None Objective lens: 25x, NA1, WD8mm two-photon excitation immersion objective lens Gel material: urethane gel Gel material consistency (1 / 4 consistency): 44 Refractive index of gel material: 1.458 Shape of gel component: truncated cone shape with top diameter 4mm, bottom diameter 18mm, and thickness 8mm Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 1100nm Container: 35mm glass bottom dish Weight: Approx. 210g Sample: HT29 cell ScalS4 cleared spheroid, approximately 350 μm thick

[0059] <Experiment 6> Observation pattern: Photographed at reference z coordinate Z = 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm Immersion fluid for comparison: None Objective lens: 25x, NA1, WD8mm two-photon excitation immersion objective lens Gel material: urethane gel Gel material consistency (1 / 4 consistency): 44 Refractive index of gel material: 1.458 Shape of gel component: truncated cone shape with top diameter 4mm, bottom diameter 18mm, and thickness 8mm Microscope: Microscope device 200 Observation method: Two-photon excitation fluorescence observation (DAPI staining) Excitation center wavelength: 700 nm (fluorescence wavelength 460-500 nm) Container: 35mm glass bottom dish Weight: Approx. 200g Sample: HT29 cell ScalS4 cleared spheroid, approximately 350 μm thick

[0060] <Experiment 7> Observation pattern: Photographed at reference z coordinate Z = 0 μm, 100 μm, 200 μm Immersion liquid and position: Silicone oil Reference z coordinate Same as gel Objective lens: 30x, NA1.05, WD0.8mm oil immersion objective lens Gel material: Silicone gel Gel material consistency (1 / 4 consistency): 111 Refractive index of gel material: 1.405 Shape of gel component: rectangular parallelepiped, 4mm long x 4mm wide x 0.9mm thick Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 1100nm Container: 384-well U-bottom microplate Weight: None Sample: HT29 cell spheroid, approximately 350 μm thick

[0061] <Experiment 8> Observation pattern: Photographed at reference z coordinate Z = 135 μm, 230 μm, 300 μm Immersion fluid compared: Silicone oil Objective lens: 30x, NA1.05, WD0.8mm oil immersion objective lens Material and position of gel material: Silicone gel Reference z coordinate Same as gel Gel material consistency (1 / 4 consistency): 111 Refractive index of gel material: 1.405 Shape of gel component: rectangular parallelepiped, 4mm long x 4mm wide x 0.9mm thick Microscope: Microscope device 200 Observation method: Two-photon excitation fluorescence observation (DAPI staining) Excitation center wavelength: 700 nm Container: 384-well U-bottom microplate Weight: Approx. 240g Sample: HT29 cell spheroid, approximately 350 μm thick

[0062] <Experiment 9> Observation pattern: Find the area of ​​interest using a 4x dry objective, then switch to a 30x immersion objective and adjust the XYZ position to take the image. Immersion fluid for comparison: None Objective lens: 4x, NA 0.16, WD 13 mm, dry objective lens 30x, NA1.05, WD0.8mm oil immersion objective lens Gel material: Silicone gel Gel material consistency (1 / 4 consistency): 69 Refractive index of gel material: 1.405 Shape of gel component: Φ6 x maximum thickness 1.1 mm (curvature 4.64 mm) spherical indentation Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 1100nm Container: 35mm glass bottom dish Weight: Approx. 130g Sample: HT29 cell spheroid, approximately 350 μm thick

[0063] <Experiment 10> Observation pattern: Search for the area of ​​interest by switching between the 4x dry objective, the 10x dry objective, and the 20x dry objective, then switch to the 30x immersion objective and take the image. Immersion fluid for comparison: None Objective lens: 4x, NA 0.16, WD 13 mm, dry objective lens 10x, NA0.3, WD10mm, dry objective lens 20x, NA0.7, WD1.7mm, dry objective lens 30x, NA1.05, WD0.8mm oil immersion objective lens Gel material: Silicone gel Gel material consistency (1 / 4 consistency): 69 Refractive index of gel material: 1.405 Shape of gel component: rectangular parallelepiped, 4mm long x 4mm wide x 0.9mm thick Microscope: Microscope device 100 Observation method: Transmitted light bright-field observation Center wavelength: 975nm Container: 35mm glass bottom dish Weight: approx. 50g Sample: HT29 cell spheroid, approximately 150 μm thick

[0064] Microscope image P3 shown in FIG. 16 is a bright-field image of a sample acquired using a 4x dry objective. Microscope image P4 shown in FIG. 17 is a bright-field image of a sample acquired using a 10x dry objective. Microscope image P5 shown in FIG. 18 is a bright-field image of a sample acquired using a 20x dry objective. Microscope image P6 shown in FIG. 19 is a bright-field image of a sample acquired using a 30x immersion objective. As shown in FIGS. 16 to 19, by using a gel member together with an immersion objective, it is possible to observe the sample in detail by gradually increasing the observation magnification without losing sight of the region of interest, even if switching from a dry objective to an immersion objective is required during observation.

[0065] <Experiment 11> Observation pattern: Move in the XY direction to switch the well to be photographed, then photograph Immersion fluid for comparison: None Objective lens: 25x, NA1, WD4mm immersion objective lens Gel material: Silicone gel Gel material consistency (1 / 4 consistency): 62 Refractive index of gel material: 1.405 Shape of gel component: truncated cone with top diameter 3mm, bottom diameter 14mm, and thickness 4mm Microscope: Microscope device 200 Observation method: Two-photon excitation fluorescence observation (DAPI staining) Excitation center wavelength: 700 nm Container: 96-well U-bottom microplate Weight: Approx. 240g Sample: HT29 cell spheroid, approximately 350 μm thick

[0066] The above-described embodiments are illustrative examples provided to facilitate understanding of the invention, and the present invention is not limited to these embodiments. Modifications and alternatives to the above-described embodiments may be included. In other words, the components of each embodiment may be modified without departing from the spirit and scope of the invention. Furthermore, new embodiments can be implemented by appropriately combining multiple components disclosed in one or more embodiments. Furthermore, some components may be deleted from or added to the components shown in each embodiment. Furthermore, the processing steps shown in each embodiment may be performed in a different order as long as they are not inconsistent. In other words, the optical signal detection device, gel member, and optical signal detection method of the present invention are susceptible to various modifications and variations without departing from the scope of the claims.

[0067] In the above-described embodiment, an example in which the optical signal detection device is an inverted microscope has been described, but the optical signal detection device is not limited to an inverted microscope. Because the gel member is not fluid, it can also be used for observation from an oblique or lateral direction. Therefore, as shown in FIG. 20 , when observing the temporal region of a relatively large animal S1, such as a mouse or marmoset, while the animal is still alive, the optical signal detection device may capture images with a gel member 310 sandwiched between an objective lens 301 tilted toward the temporal region of the animal S1 and the temporal region of the animal S1. In this case, a cover glass C1 has been implanted in the temporal region of the animal S1 by prior surgery, and the space between the cover glass C1 and the objective lens 301 may be filled with the gel member 310.

[0068] In the above-described embodiment, a gel member is first attached to the objective lens, and then the objective lens is brought close to the container, filling the space between the container and the objective lens with the gel member. However, the procedure for attaching the gel member is not particularly limited as long as the gel member fills the space between the container and the objective lens. For example, as shown in FIGS. 21 and 22, observation may be performed using a gel member 410 attached to the backside of a microplate C2 having multiple wells. The gel member 410 preferably has a ¼ consistency of 44 to 111, and deforms to fit the shape of the backside of the microplate C2, thereby adhering tightly to the backside of the microplate C2. In this case, pressing the objective lens against the gel member 410 on the backside of the microplate C2 fills the space between the objective lens and the microplate C2 with the gel member 410, making it easy to observe samples with a high numerical aperture.

[0069] In the above-described embodiment, an existing immersion objective lens designed for immersion media such as silicone oil or water is used, and a gel material is used instead of the immersion medium. However, it is also possible to use a newly designed objective lens that matches the gel material to be used. This allows a new objective lens to be designed to match the refractive index of the expected gel material, increasing the freedom of selection of the gel material. [Explanation of symbols]

[0070] 1, 100, 200 Microscope equipment 3, 107, 206, 301 Objective Lenses 6 1 / 4 cone 7 Test Object 10~12, 310, 410 Gel material C container C1 cover glass C2 Microplate L distance P1~P6 Microscope images S sample S1 animals

Claims

1. An objective lens, a holding member for holding the sample between the objective lens and the sample; a silicone gel filling a space between the objective lens and the holding member, The silicone gel has a 1 / 4 consistency of 44 or more and 111 or less, as measured based on a consistency test using a 1 / 4 cone according to JIS K 2220. An optical signal detection device characterized by:

2. 2. The optical signal detection device according to claim 1, The contact area of ​​the silicone gel with the objective lens is larger than the contact area of ​​the silicone gel with the holding member. An optical signal detection device characterized by:

3. 3. The optical signal detection device according to claim 1, further comprising: a revolver to which the objective lens is attached, When the revolver rotates, the silicone gel adsorbed to the objective lens moves together with the objective lens out of the optical path of the objective lens. An optical signal detection device characterized by:

4. 4. The optical signal detection device according to claim 1, The silicone gel is removably attached to the objective lens. An optical signal detection device characterized by:

5. 5. The optical signal detection device according to claim 1, The silicone gel has a tapered shape in which the cross-sectional area decreases from the objective lens toward the holding member. An optical signal detection device characterized by:

6. 6. The optical signal detection device according to claim 5, The silicone gel has a truncated cone shape. An optical signal detection device characterized by:

7. 6. The optical signal detection device according to claim 1, The silicone gel has a convex surface formed by a curved surface on the holding member side. An optical signal detection device characterized by:

8. 8. The optical signal detection device according to claim 1, The thickness of the silicone gel is 1.1 times or more and 1.5 times or less the working distance of the objective lens. An optical signal detection device characterized by:

9. 9. The optical signal detection device according to claim 1, The difference in refractive index between the silicone gel and the holding member is within ±0.

1. An optical signal detection device characterized by:

10. 10. The optical signal detection device according to claim 1, The difference in refractive index between the silicone gel and the sample or the medium surrounding the sample is within ±0.

1. An optical signal detection device characterized by:

11. Illuminating the sample in a state where a space between the objective lens and a holding member for holding the sample is filled with a silicone gel having a 1 / 4 consistency of 44 or more and 111 or less as measured based on a consistency test using a 1 / 4 cone according to JIS K 2220; and detecting, with a photodetector, light from the sample that has entered through the objective lens.

1. A method for detecting an optical signal, comprising:

12. The optical signal detection method according to claim 11, further comprising: changing a distance between the objective lens and the sample in the optical axis direction of the objective lens; and repeating the steps of illuminating the sample and detecting light from the sample with a photodetector for each change in the distance.

1. A method for detecting an optical signal, comprising:

13. 11. The optical signal detection device according to claim 1, The objective lens is an immersion objective lens. An optical signal detection device characterized by:

14. 13. The optical signal detection method according to claim 11 or 12, The objective lens is an immersion objective lens.

1. A method for detecting an optical signal, comprising:

Images