JP2022071821A - Light signal detector, gel member, and method for detecting light signal - Google Patents

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

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

Currently, research using cell aggregates such as spheroids and organoids, in which a large number of cells are collected and cultured three-dimensionally, is drawing attention. The sample as described above has a size of, for example, about 100 μm to 500 μm.

In deep cell observation of such a sample, an immersion objective lens is generally used. In an immersion objective lens, it is possible to realize a higher numerical aperture than a dry objective lens by filling the space between the objective lens and the sample (more precisely, the holding member that holds the sample) with immersion liquid. be.

By using an immersion liquid having a refractive index close to that of the sample, it is possible to suppress spherical aberration caused by the refractive index mismatch that occurs at the interface between the sample and the immersion liquid (air in the case of a dry objective lens). Is. As described in Patent Document 1 and the like, the effect of spherical aberration due to the refractive index mismatch becomes more pronounced as the observation position becomes deeper, so it is possible to observe deeper positions by suppressing spherical aberration. Become.

Japanese Unexamined Patent Publication No. 2017-0266666

By the way, in actual observation, the dry objective lens and the immersion objective lens may be switched and used in the middle of observation. For example, first, a dry objective lens with a relatively low magnification and a wide field of view is used to search for the region of interest, and then an immersion objective lens with high resolution is used to observe the region of interest in detail. , A typical example.

However, when switching from the dry objective lens to the immersion objective lens, it is necessary to newly supply the immersion liquid and fill the space between the objective lens and the sample with the immersion liquid. Further, when switching from the immersion objective lens to the dry objective lens, it is necessary to surely wipe off the immersion liquid so that the immersion liquid does not remain on the sample surface. As described above, in the switching between the dry objective lens and the immersion objective lens, various operations other than the switching work of the objective lens itself are additionally required. Therefore, the observation is temporarily interrupted, and smooth observation becomes difficult.

Further, even when observing the sample only with the immersion objective lens, there are various problems as compared with the case where the dry objective lens is used. For example, when water is used as the immersion liquid, the immersion liquid evaporates if the observation is continued for a long time. Therefore, it is necessary to appropriately supply the immersion liquid during the observation. Further, when oil is used as the immersion liquid, there are problems that cleaning is troublesome and bubbles are likely to be generated due to high viscosity. Further, when an objective lens having a long working distance is used in an inverted microscope, it becomes difficult to maintain the immersion liquid between the objective lens and the sample by surface tension. Even when observing the sample from an oblique direction or a lateral direction, it is difficult to maintain the immersion liquid by surface tension, and in that respect, there is a problem similar to that when an objective lens having a long working distance is used. Therefore, in these cases, a large-scale mechanism for maintaining the immersion liquid between the objective lens and the sample is required. Further, when observing over a wide range in the depth direction, there is a problem that the immersion liquid easily spills from between the objective lens and the sample because the distance between the objective lens and the sample changes greatly during the observation.

As described above, at present, when an immersion objective lens is used, a greater burden is imposed on the user than when a dry objective lens is used. Based on the above circumstances, an object of one aspect of the present invention is to provide a technique capable of good observation using an immersion objective lens while reducing the burden on the user.

The optical signal detection device according to an embodiment of the present invention is a gel member that fills the space between the objective lens, the holding member that holds the sample between the objective lens and the sample, and the space between the objective lens and the holding member. The gel member has a 1/4 consistency indicating a value of 44 or more and 111 or less, which is measured based on a consistency test using a 1/4 cone of JIS K 2220.

The gel member according to the embodiment of the present invention is a gel member attached to the tip of an objective lens, and the gel member is measured based on a consistency test using a 1/4 cone of JIS K 222044. It has a 1/4 consistency showing a value of 111 or less.

In the optical signal detection method according to the embodiment of the present invention, the space between the objective lens and the holding member for holding the sample is measured based on the consistency test using a 1/4 cone of JIS K 222044. The sample is illuminated with the gel member having a 1/4 consistency showing a value of 111 or less, and the light from the sample incident via the objective lens is detected by the light detector. Including that.

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.

It is a figure which illustrated the structure of the microscope apparatus which concerns on one Embodiment. It is a figure for demonstrating the consistency measurement method. It is a figure for demonstrating the shape of a gel member. It is a figure which showed an example of the shape of a gel member. It is a figure which showed another example of the shape of a gel member. It is a figure which showed still another example of the shape of a gel member. It is a figure for demonstrating the method of attaching and detaching the gel member of an objective lens. It is a figure which made an example of the relationship between a sample and a medium in a container. It is a figure which showed another example of the relationship between a sample and a medium in a container. It is a table which shows the result of having compared the case where the oil was used as the immersion liquid, and the case where the gel member was used. It is another table which shows the result of having compared the case where the oil was used as the immersion liquid, and the case where the gel member was used. It is a figure explaining the structure of the microscope apparatus used in an experiment. It is a figure explaining the structure of another microscope apparatus used for an experiment. It is a microscope image obtained using oil as an immersion liquid in Experiment 1. It is a microscope image obtained by using a gel member as an immersion liquid in Experiment 1. It is a microscope image acquired using the 4x dry objective lens in Experiment 10. It is a microscope image acquired by using a 10 times dry objective lens in Experiment 10. It is a microscope image acquired by using a 20x dry objective lens in Experiment 10. It is a microscope image acquired using the immersion system objective lens of 30 times in Experiment 10. It is a figure which showed the observation example from an oblique direction. It is a perspective view of a microplate. It is a figure for demonstrating the structure of a microplate.

FIG. 1 is a diagram illustrating the configuration of the microscope device according to the embodiment. The microscope device 1 shown in FIG. 1 is an inverted microscope for observing the sample S from below, and is an example of an optical signal detection device for detecting an optical signal. As shown in FIG. 1, the microscope device 1 includes a light source 2, an objective lens 3 mounted on the revolver 4, and a camera 5. The objective lens 3 is a so-called immersion type objective lens used in a state where the space between the objective lens 3 and the container is filled with an immersion liquid. The container C for accommodating the sample S is, for example, a glass bottom dish or the like, and is an example of a holding member for holding the sample S. The sample S is not particularly limited, but is, for example, a biological sample such as a cell. The sample S may be a sample having a thickness of several hundred μm, such as a three-dimensionally cultured spheroid or organoid.

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

The microscope device 1 irradiates the sample S with the light from the light source 2, and detects the light from the sample S with the camera 5 to acquire an image of the sample S. The observation method of the microscope device 1 is not particularly limited. For example, the image of the sample S may be acquired by the bright field observation method, or the image of the sample S may be acquired by the fluorescence observation method. The observation method may be another observation method, a phase difference observation method, a differential interference contrast observation method, or the like.

The microscope device 1 includes a gel member 10 having a refractive index higher than that of air between the immersion objective lens 3 and the container C instead of the liquid immersion liquid.

The gel member 10 has no fluidity unlike the immersion liquid. Therefore, the gel member 10 can be easily arranged in the observation optical path between the objective lens 3 and the container C as compared with the immersion liquid held between the objective lens 3 and the container C by using surface tension. Therefore, the work of removing it from the observation optical path is also easy. Therefore, even when the immersion type objective lens and the dry type objective lens are switched and used, the switching operation can be performed quickly. Further, the gel member 10 having no fluidity does not flow down from the objective lens 3 during observation unlike the immersion liquid. Therefore, the objective lens 3 and its surroundings are not soiled, and therefore cleaning after use is easy. Further, since the volume does not decrease extremely due to evaporation, it is not necessary to additionally supply the volume during observation unlike immersion, and it is possible to easily cope with long-term observation. Further, since the gel member 10 has a relatively high adhesiveness, it can stably exist between the objective lens 3 and the container C even when observed obliquely or from the side. As described above, the gel member 10 is much easier to handle than the immersion objective lens, and the burden on the user due to the use of the immersion objective lens can be significantly reduced.

Further, the gel member 10 has a higher refractive index than air, like the immersion liquid. Therefore, it is possible to realize a high numerical aperture by demonstrating the performance of the immersion type objective lens 3, and it is possible to obtain a bright image with high resolution. In addition, the difference in refractive index from the container C and the sample S is smaller than when air is present. Therefore, as in the case of using immersion liquid, the sample S can be observed deeply while suppressing the spherical aberration caused by the difference in the refractive index from the container C and the sample S. Therefore, by using the gel member 10, the three-dimensional structure of the sample can be satisfactorily observed.

As described above, according to the microscope device 1 for observing using the gel member 10 instead of the immersion liquid, even when the immersion type objective lens 3 is used, the user is forced to bear an excessive burden. The sample S can be observed well without any problem. Further, since the switching between the dry objective lens and the immersion objective lens can be smoothly performed, the interruption of observation due to the switching of the objective lens can be suppressed in a short time and the observation can be performed efficiently.

FIG. 2 is a diagram for explaining a consistency measuring method. FIG. 3 is a diagram for explaining the shape of the gel member. 4 to 6 are views showing an example of the shape of the gel member. FIG. 7 is a diagram for explaining a method of attaching and detaching the gel member of the objective lens. Hereinafter, a desirable configuration of the microscope device 1 will be described with reference to FIGS. 2 to 7.

It is desirable that the gel member 10 included in the microscope device 1 has a 1/4 consistency indicating a value of 44 or more and 111 or less, which is measured based on the consistency test using the 1/4 cone of JIS K 2220 described above. In general, the consistency, as defined in JIS K 2220, refers to the distance that the standard cone and the option cone enter the sample under the specified conditions of load, time and temperature, and is a numerical value measured in units of 0.1 mm. Is multiplied by 10. On the other hand, 1/4 consistency is 1/4 consistency (one quarter scale penetration) defined in JIS K 2220, and is a standard cone or a specified cone obtained by reducing an optional cone to 1/4 (1/4 cone). ) Is measured. That is, as shown in FIG. 2, the distance L at which the 1/4 cone 6 enters the test object 7 under the specified conditions of load, time, and temperature is referred to, and the value measured in units of 0.1 mm is multiplied by 10. It represents. The measurement is performed under a temperature condition of 25 ° C. ± 0.5 ° C.

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 in place of immersion. Therefore, it becomes difficult for the gel member 10 to maintain its own shape, and the gel member 10 is crushed. As a result, the space between the objective lens 3 and the container C cannot be filled with the gel member 10, and good observation may not be possible. In particular, when the sample S is observed with the objective lens 3 having a long working distance (for example, WD = 4 mm) and the distance between the objective lens 3 and the container C being long, the above-mentioned situation is likely to occur. Further, if the gel member 10 is too soft, when the gel member 10 having a predetermined shape is produced using a mold, the shape cannot be maintained when the gel member 10 is peeled from the mold, and the proportion of defective products increases. Resulting in. In addition, the durability is low, which limits the number of times it can be used repeatedly.

On the other hand, when the 1/4 consistency of the gel member 10 is less than the lower limit value 44, the gel member 10 becomes too hard for use in place of immersion liquid. When the gel member 10 has an appropriate hardness, the gel member 10 is deformed according to the distance between the objective lens 3 and the container C, so that excessive pressure is not applied to the container C. The space between the objective lens 3 and the container C can be appropriately filled with the gel member 10. On the other hand, when the gel member 10 is too hard, that is, when the 1/4 consistency of the gel member 10 is less than the lower limit value 44, the distance between the objective lens 3 and the container C changes. However, the gel member 10 does not flexibly deform. Therefore, for example, when the objective lens 3 is brought close to the container C for deep observation, the gel member 10 pressed by the objective lens 3 does not sufficiently spread between the objective lens 3 and the container C, and the objective lens 3 is used. The distance between the container C and the container C does not shrink to the expected distance. For this reason, a large force is applied to the container C, which may cause inconveniences such as deformation of the container and displacement of the position of the container. As a result, the observation position also shifts, making it difficult to correctly observe the region of interest.

Even when the distance between the objective lens 3 and the container C is variable because the 1/4 consistency of the gel member 10 is 44 or more and 111 or less, the microscope device 1 is suitable without causing any inconvenience. It is possible to make observations. Further, even when the positional relationship between the objective lens 3 and the container C changes in the direction orthogonal to the optical axis, the microscope device 1 can appropriately observe without causing any inconvenience. Therefore, according to the microscope device 1, the effect of satisfactorily observing the sample S without imposing an excessive burden on the user can be maintained over a wide observation range.

In the gel member 10 that fills the space between the objective lens 3 and the container C, as shown in FIG. 3, the contact area of the gel member 10 with the objective lens 3 (hereinafter referred to as the first contact area) is the gel. It is desirable that the member 10 has a larger contact area with the container C (hereinafter referred to as a second contact area). Therefore, for example, the gel member 10 may have a truncated cone shape as shown in FIG. 4, and in that case, it is desirable that the gel member 10 is arranged with the apex of the cone facing the objective lens 3 side. Note that FIG. 3 shows that the gel member 10 is in contact with the container C in a circular region having a radius R1 and the gel member 10 is in contact with the objective lens 3 in a circular region having a radius R2 (> radius R1). It is shown.

Since the first contact area is larger than the second contact area, the gel member 10 can be more firmly attached to the objective lens 3 than the container C. As a result, when the distance between the objective lens 3 and the container C is widened to be equal to or larger than the thickness of the gel member 10, the gel member 10 can be peeled off from the container C and stay in the objective lens 3. Therefore, according to the microscope device 1, when the objective lens 3 is switched to another objective lens, the gel member 10 adsorbed on the objective lens 3 moves out of the optical path together with the objective lens 3 due to the rotation of the revolver, so that the gel member 10 It is possible to omit the user's work of removing the objective lens, and the objective lens can be switched smoothly.

In FIG. 4, the gel member 10 having a truncated cone shape is illustrated, but the shape of the gel member included in the microscope device 1 is not limited to the shape of a truncated cone like the gel member 10. For example, instead of the gel member 10, the microscope device 1 may include a cylindrical gel member 11 as shown in FIG. 5, or may include a prism-shaped or pyramidal-shaped gel member.

Further, the microscope device 1 may include a gel member 12 including a bottom surface and a convex surface formed by a curved surface as shown in FIG. 6, in which case the gel member 12 may have a convex surface on the container C side. desirable. As shown in the gel member 10 and the gel member 12, the gel member has a tapered shape in which the cross-sectional area decreases from the objective lens 3 toward the container C, so that the above-mentioned magnitude relationship between the contact areas can be easily realized. Can be done. Therefore, from the viewpoint of simplifying the switching work of the objective lens, 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.

As described above, the gel member has adhesiveness and is detachably attached to the objective lens. Therefore, when attaching the gel member to the objective lens 3, as shown in FIG. 7, the gel member (here, the gel member 12) is simply placed on the tip lens 3a portion of the objective lens 3 using tweezers or the like. It's fine. As a result, the gel member 12 is attracted to the objective lens 3, so that the gel member 12 can be easily attached to the objective lens 3. Similarly, tweezers or the like may be used when the gel member 12 is removed from the objective lens 3. As described above, since the gel member 12 is detachably attached to the objective lens 3, the preparatory work for observation can be easily performed.

Hereinafter, a more desirable configuration of the microscope device 1 will be described. It is desirable that the thickness of the gel member (for example, the gel member 10) included in the microscope device 1 is 1.1 times or more and 1.5 times or less the working distance of the objective lens 3. Since the thickness of the gel member 10 is 1.1 times or more the working distance, even if the thickness of the container C is taken into consideration, the space between the container C and the objective lens 3 is filled with the gel member 10 without a gap. The surface of the sample S (the bottom surface of the sample S in FIG. 1) can be observed. Further, since the thickness of the gel member 10 is 1.5 times or less the working distance, it is possible to prevent the gel member 10 from being excessively compressed when observing the deep part of the sample S. If the gel member 10 is compressed too much, a large pressure is applied to the container C, and the container C is deformed or the position of the container C is displaced. As a result, the observation position shifts, making it difficult to correctly observe the region of interest. If the working distance is 1.5 times or less, the pressure on the gel member 10 is not significantly applied, so that the deformation of the container C and the misalignment of the container C are reduced so that the observation can be performed at the intended position. Become. Therefore, even in the Z-stack imaging performed while scanning the sample S in the depth direction, it is possible to acquire an image at an intended position while filling the space between the container C and the objective lens 3 with the gel member 10 without gaps.

Further, it is desirable that the difference in refractive index between the gel member 10 and the container C is within ± 0.1. By suppressing the difference in refractive index within ± 0.1, spherical aberration caused by the refractive index mismatch can be suppressed and good observation can be performed. Since the effect is remarkable in deep observation, it is particularly suitable for observing a thick sample. The standard refractive index of the bottom surface of the glass bottom dish and the cover glass is 1.52. Therefore, when the thickness of the glass bottom dish or the cover glass varies widely, the gel member 10 may have a refractive index in the range of, for example, 1.52 ± 0.1. As a result, spherical aberration generated by a change in thickness can be reduced without using the correction ring mechanism of the objective lens.

Further, it is desirable that the difference in refractive index between the gel member 10 and the sample S or the medium M covering the sample S is within ± 0.1. 8 and 9 are diagrams illustrating the relationship between the sample and the medium in the container. The medium M is not particularly limited, but is, for example, a culture solution, a clearing solution, or the like. By suppressing the difference in refractive index within ± 0.1, spherical aberration caused by the refractive index mismatch that occurs in deep observation can be suppressed and good observation can be performed. 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, if the refractive index difference is within ± 0.1, it is caused by the refractive index mismatch that occurs in the observation of the sample S through the medium M. It is possible to observe the sample S satisfactorily while suppressing the spherical aberration.

10 and 11 are tables showing the results of comparison between the case where oil is used as the immersion liquid and the case where the gel member is used. Hereinafter, with reference to FIGS. 10 and 11, a comparison between the case of using a gel member having a quarter consistency in the range of 44 or more and 111 or less, which was found as a desirable range by the inventor of the present application, and the case of using oil. The results will be described.

Table T1 shown in FIG. 10 and T2 shown in FIG. 11 are reference z-coordinates (Z) with reference to the bottom surface of the sample S when observing with oil, and observation z-coordinates (Z coordinates) indicating the Z-coordinate of the microscope device at that time. The relationship of P) and the relationship between the reference z-coordinate (Z) and the observation z-coordinate (P) when observing using the gel member are shown. The unit of the z coordinate is μm. It should be noted that the combination of Z and P when the gel member was used was obtained so as to be close to the image obtained by the combination of Z and P when oil was used, to the extent that it can be judged to be substantially the same. It is a combination of Z and P of time. Further, the results of FIGS. 10 and 11 are the results obtained by using the microscope of FIG. 12 described later.

The gel members used are five types of gel members having a consistency of 55, 62, 69, 91, and 111. It should be noted that the consistency here is 1/4. Table T1 shows that the z-stack imaging was performed 6 times on the gel member (gel number 1) having a consistency of 55. In addition, z-stack photography was performed 6 times, 4 times, and 5 times with the gel member with a consistency of 62 (gel number 2), the gel member with a consistency of 69 (gel number 3), and the gel member with a consistency of 91 (gel number 4), respectively. It is shown that it was done once. Further, Table T2 shows that four gel members having a consistency of 111 were prepared (gel numbers 5 to 8), and z-stack photography was performed twice for each.

First, pay attention to the result of the gel member (gel number 1) having the consistency 55 in Table T1. The result of the first z-stack photography is excluded from the examination because the conditions are significantly different from those of the second and subsequent z-stack photography in that the gel member is a result in a state where the gel member has not been crushed yet. Consider the results from the second time onward. In the first z-stack imaging, positions that are the same as the images at depths of 0 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, and 300 μm when oil is used are measured.

Focusing on the results of the second and subsequent times of the gel member having a consistency of 55 (gel number 1), particularly the observation z position at which the image corresponding to the depth of 200 μm in the oil was acquired, the variation of the observation z position is 7 μm at the maximum. On the other hand, a gel member having a consistency of 62 (gel number 2), a gel member having a consistency of 69 (gel number 3), a gel member having a consistency of 91 (gel number 4), and a gel member having a consistency of 111 (gel number 3). In 5-8), the variation of the observed z position is 5 μm, 3 μm, 2 μm, and 1 μm, respectively.

From this result, it can be seen that the variation in the observation position increases as the consistency is lower, that is, as the gel member is harder. This is because the harder the gel member, the greater the pressure applied to the container or sample when the gel member deforms into a shape corresponding to the distance between the objective lens and the container, and as a result, the position of the container or sample changes. It is presumed that it was done. On the other hand, assuming that the size of the cell is 20 μm, if the variation in the observation position can be suppressed to 10 μm, which is half of that, it is possible to capture and observe the same cell when repeated observation is performed. Therefore, from the measurement results shown in FIGS. 10 and 11, it can be determined that the variation can be suppressed to 10 μm if it is 44 or more, which is the lower limit value of the above-mentioned desirable 1/4 consistency range. That is, the reproducibility of the observation position can be ensured by satisfying the above-mentioned desirable 1/4 consistency range of the gel member.

Next, pay attention to the results in Table T2. This is the result of Z-stack photography performed twice for each of the four gel members having the same consistency 111. Although the description is omitted in Table T2, the Z position is stable, and except for gel number 7, the result of the same Z position as the second Z stack photography of each gel is obtained even in the third Z stack photography. became. However, gel number 7 could not be photographed for the third time in the shallow part of the sample. The gel is crushed during the second deep region observation, the gel shape is not restored normally during the third shallow region observation, and the space between the container and the objective lens cannot be filled with the gel member. This is because the.

From the above results, it may happen that the gel with a consistency of 111 is not restored because the gel is crushed and the observation of the shallow region of the sample cannot be repeated, but 75% of the gels should be repeatedly observed. Therefore, it can be said that the gel member can be used repeatedly if the consistency 111 is set as the upper limit value. Therefore, by satisfying the above-mentioned desirable 1/4 consistency range of the gel member, the gel member can change its shape according to the distance between the container and the objective lens even when it is used repeatedly. It is possible and can fill the space between the container and the objective lens.

As described above, it is desirable that the gel member has a 1/4 consistency showing a value of 44 or more and 111 or less.

12 and 13 are diagrams illustrating the configuration of the microscope device used in the experiment. FIG. 14 is a microscope image obtained by using oil as an immersion liquid in Experiment 1, and FIG. 15 is a microscope image obtained by using a gel member as an immersion liquid in Experiment 1. 16 to 18 are microscope images obtained in Experiment 10 using 4x, 10x, and 20x dry objective lenses, respectively. FIG. 19 is a microscope image obtained in Experiment 10 using a 30x immersion objective lens.

Hereinafter, various experiments conducted by the inventor will be described with reference to FIGS. 12 to 19, and the results will be considered. First, the microscope device used in the experiment will be described with reference to FIGS. 12 and 13.

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

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

The microscope device 100 acquires an image by a bright field observation method. Specifically, the microscope device 100 is a bandpass filter in a state where the space between the objective lens 107 and the container C is filled with a gel member 10 having a 1/4 consistency showing a value of 44 or more and 111 or less. The image of the sample S is acquired by irradiating the sample S with light in the near-infrared region that has passed through the 102 and is difficult to scatter, and detecting the light from the sample S incident through the objective lens 107 with the camera 109. Further, the microscope device 100 changes the distance in the optical axis direction between the objective lens 107 and the sample S (container C), and each time the distance is changed, the sample S is irradiated with light and the light from the sample S is detected. May be repeated.

The microscope device 200 shown in FIG. 13 is an inverted microscope device, more specifically, a two-photon excitation laser scanning microscope device, which is an example of an optical signal detection device for detecting an optical signal. The microscope device 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 illuminated optical path from the laser 201 to the sample S. The scanner 202 is a two-dimensional scanner that deflects the illumination light in two directions orthogonal to the optical axis of the objective lens 206, and may include, for example, a galvanometer scanner and a resonant scanner. Further, the microscope device 200 includes a light detector 208, and the objective lens 206, the dichroic mirror 205, and the objective lens 206 are provided on the observation optical path from the sample S held in the container C to the light detector 208. It includes a relay lens 207 that relays the pupil to the light detector 208.

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

The microscope device 200 acquires an image by a two-photon excitation fluorescence observation method. Specifically, the microscope device 200 uses a 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 1/4 consistency showing a value of 44 or more and 111 or less. The light detector 208 is obtained by irradiating the sample S with a laser beam having an excitation wavelength of 700 nm while moving the irradiation position in two dimensions, and detecting the fluorescence from the sample S incident via the objective lens 206 with the objective lens 206. The image of the sample S is acquired based on the signal detected in 1 and the information of the irradiation position. Further, the microscope device 200 changes the distance in the optical axis direction between the objective lens 206 and the sample S (container C), and each time the distance is changed, the sample S is irradiated with light and the fluorescence from the sample S is detected. May be repeated.

There were a total of 11 types of experiments, from Experiment 1 to Experiment 11, and it was confirmed that good observation was performed using the gel member. Regarding whether or not good observation was performed, in Experiments 1-4 and 7-8, the image obtained by using the gel member and the image obtained by immersion liquid such as water or oil were used. Confirmed by comparison. Further, for Experiments 5-6 and 9-11, the images obtained by using the gel member were confirmed alone. The details of each experiment are as follows.

<Experiment 1>
Observation pattern: Photographed at the reference z-coordinate Z = 52 μm, 156 μm, 263 μm (photographed at the z-coordinate position where an image similar to the image taken at each reference z-coordinate position of the immersion liquid to be compared is acquired)
Comparison target immersion liquid and position: Silicone oil Reference z coordinate Z = 50 μm, 150 μm, 250 μm
Objective lens: 30x, NA 1.05, WD 0.8mm oil-immersed objective lens Gel member material: Silicone gel Gel member consistency (1/4 consistency): 69
Refractive index of gel member: 1.405
Shape of gel member: rectangular parallelepiped shape of length 4 x width 4 x thickness 0.9 mm Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 1100 nm
Container: 35mm glass bottom dish Weight: Approximately 210g
Sample: HT29 cell spheroid, about 350 μm thick

The microscope image P1 shown in FIG. 14 is a bright field image of a sample obtained by using silicone oil at Z = 150 μm. The microscope image P2 shown in FIG. 15 is a bright field image of a sample obtained by using a gel member at Z = 156 μm. From FIGS. 14 and 15, it can be confirmed that an image comparable to that when silicone oil is used can be obtained even when the gel member is used.

<Experiment 2>
Observation pattern: Photographed at reference z-coordinate Z = 51 μm, 152 μm, 255 μm (photographed at z-coordinate position where an image similar to the image taken at each reference z-coordinate position of the immersion liquid to be compared is acquired)
Comparison target immersion liquid and position: Silicone oil Reference z coordinate Z = 50 μm, 150 μm, 250 μm
Objective lens: 30x, NA 1.05, WD 0.8mm oil-immersed objective lens Gel member material: Silicone gel Gel member consistency (1/4 consistency): 111
Refractive index of gel member: 1.405
Shape of gel member: rectangular parallelepiped shape of length 4 x width 4 x thickness 0.9 mm Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 1100 nm
Container: 35mm glass bottom dish Weight: Approximately 210g
Sample: HT29 cell spheroid, about 350 μm thick

<Experiment 3>
Observation pattern: Photographed at the reference z-coordinate Z = 52 μm, 156 μm, 260 μm (taken at the z-coordinate position where an image similar to the image taken at each reference z-coordinate position of the immersion liquid to be compared is acquired)
Comparison target immersion liquid and position: Silicone oil Reference z coordinate Z = 50 μm, 150 μm, 250 μm
Objective lens: 30x, NA 1.05, WD 0.8mm oil-immersed objective lens Gel member material: Silicone gel Gel member consistency (1/4 consistency): 69
Refractive index of gel member: 1.405
Shape of gel member: Φ6 x maximum thickness 1.1 mm (curvature 4.64 mm) missing ball Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 1100 nm
Container: 35mm glass bottom dish Weight: Approximately 210g
Sample: HT29 cell spheroid, about 350 μm thick

<Experiment 4>
Observation pattern: Photographed at the reference z-coordinate Z = 49 μm, 148 μm, 246 μm (photographed at the z-coordinate position where an image similar to the image taken at each reference z-coordinate position of the immersion liquid to be compared is acquired)
Comparison target immersion liquid and position: water reference z coordinate Z = 49 μm, 149 μm, 245 μm
Objective lens: 25x, NA1.05, WD2mm water immersion objective lens Gel member material: Aqua joint Gel member consistency (1/4 consistency): 96
Refractive index of gel member: 1.356
Shape of gel member: Top surface Φ3 x Bottom surface Φ7 x 2.5 mm thick cone shape Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 1100 nm
Container: 35mm glass bottom dish Weight: Approximately 130g
Sample: HT29 cell spheroid, about 350 μm thick

<Experiment 5>
Observation pattern: Photographed at reference z coordinate Z = 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm Comparison target immersion: None Objective lens: 25x, NA1, WD8 mm Two-photon excitation immersion objective lens Gel member material: Urethane gel Consistency of gel member (1/4 consistency): 44
Refractive index of gel member: 1.458
Shape of gel member: Top surface Φ4 × Bottom surface Φ18 × Thickness 8 mm truncated cone shape Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 1100 nm
Container: 35mm glass bottom dish Weight: Approximately 210g
Sample: HT29 cell ScalS4 clearing spheroid, about 350 μm thick

<Experiment 6>
Observation pattern: Photographed at reference z coordinate Z = 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm Comparison target immersion: None Objective lens: 25x, NA1, WD8 mm Two-photon excitation immersion objective lens Gel member material: Urethane gel Consistency of gel member (1/4 consistency): 44
Refractive index of gel member: 1.458
Shape of gel member: Top surface Φ4 × Bottom surface Φ18 × Thickness 8 mm truncated cone shape 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: Approximately 200g
Sample: HT29 cell ScalS4 clearing spheroid, about 350 μm thick

<Experiment 7>
Observation pattern: Photographed at reference z-coordinate Z = 0 μm, 100 μm, 200 μm Comparison target immersion liquid and position: Silicone oil Same as reference z-coordinate gel Objective lens: 30 times, NA 1.05, WD 0.8 mm oil immersion objective lens Gel member Material: Silicone gel Consistency of gel member (1/4 consistency): 111
Refractive index of gel member: 1.405
Shape of gel member: rectangular parallelepiped shape of length 4 x width 4 x thickness 0.9 mm Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 1100 nm
Container: 384-hole U-bottom microplate Weight: None Sample: HT29 cell spheroid, thickness approx. 350 μm

<Experiment 8>
Observation pattern: Photographed at reference z coordinate Z = 135 μm, 230 μm, 300 μm Comparison target immersion liquid: Silicone oil Objective lens: 30 times, NA1.05, WD 0.8 mm oil immersion objective lens Gel member material and position: Silicone gel reference z coordinate Same as gel Consistency of gel member (1/4 consistency): 111
Refractive index of gel member: 1.405
Shape of gel member: rectangular parallelepiped shape of length 4 x width 4 x thickness 0.9 mm Microscope: Microscope device 200
Observation method: Two-photon excitation fluorescence observation (DAPI staining)
Excitation center wavelength: 700 nm
Container: 384-hole U-bottom microplate Weight: Approximately 240 g
Sample: HT29 cell spheroid, about 350 μm thick

<Experiment 9>
Observation pattern: Search for a region of interest with a 4x dry objective lens, switch to a 30x immersion objective lens and adjust the XYZ position for shooting. Comparison target immersion: None Objective lens: 4x, NA0.16, WD13mm, dry System objective lens 30x, NA 1.05, WD 0.8mm oil-immersed objective lens Gel member material: Silicone gel Gel member consistency (1/4 consistency): 69
Refractive index of gel member: 1.405
Shape of gel member: Φ6 x maximum thickness 1.1 mm (curvature 4.64 mm) missing ball Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 1100 nm
Container: 35mm glass bottom dish Weight: Approximately 130g
Sample: HT29 cell spheroid, about 350 μm thick

<Experiment 10>
Observation pattern: Switch to the 4x dry objective lens, 10x dry objective lens, and 20x dry objective lens in this order to search for the area of interest, and then switch to the 30x immersion objective lens to take a picture. None Objective lens: 4x, NA0.16, WD13mm, dry objective lens
10x, NA0.3, WD10mm, dry objective lens
20x, NA0.7, WD1.7mm, dry objective lens
Oil immersion objective lens with 30x, NA1.05, WD0.8mm Gel member material: Silicone gel Gel member consistency (1/4 consistency): 69
Refractive index of gel member: 1.405
Shape of gel member: rectangular parallelepiped shape of length 4 x width 4 x thickness 0.9 mm Microscope: Microscope device 100
Observation method: Transmission bright field observation Center wavelength: 975 nm
Container: 35mm glass bottom dish Weight: Approximately 50g
Sample: HT29 cell spheroid, about 150 μm thick

The microscope image P3 shown in FIG. 16 is a bright-field image of a sample obtained by using a 4x dry objective lens. The microscope image P4 shown in FIG. 17 is a bright-field image of a sample obtained by using a 10x dry objective lens. The microscope image P5 shown in FIG. 18 is a bright-field image of a sample obtained by using a 20x dry objective lens. The microscope image P6 shown in FIG. 19 is a bright field image of a sample obtained by using a 30x immersion objective lens. As shown in FIGS. 16 to 19, by using the gel member together with the immersion objective lens, even when the switching from the dry objective lens to the immersion objective lens is included during the observation, the region of interest It is possible to observe the sample in detail while gradually increasing the observation magnification without losing sight of it.

<Experiment 11>
Observation pattern: Move in the XY direction to switch the well to be photographed, and then perform the imaging comparison subject Immersion: None Objective lens: 25x, NA1, WD4mm immersion objective lens Gel member material: Silicone gel Consistency (1/4 consistency): 62
Refractive index of gel member: 1.405
Shape of gel member: Top surface Φ3 × Bottom surface Φ14 × Thickness 4 mm truncated cone shape Microscope: Microscope device 200
Observation method: Two-photon excitation fluorescence observation (DAPI staining)
Excitation center wavelength: 700 nm
Container: 96-hole U-bottom microplate Weight: Approximately 240 g
Sample: HT29 cell spheroid, about 350 μm thick

The above-described embodiments show specific examples for facilitating the understanding of the invention, and the present invention is not limited to these embodiments. Modifications of the above embodiments and alternatives to the above embodiments may be included. That is, each embodiment can modify the components within a range that does not deviate from the purpose and scope. In addition, a new embodiment can be implemented by appropriately combining a plurality of components disclosed in one or more embodiments. In addition, some components may be deleted from the components shown in each embodiment, or some components may be added to the components shown in the embodiments. Further, the processing procedures shown in each embodiment may be performed in a different order as long as there is no contradiction. That is, the optical signal detection device, the gel member, and the optical signal detection method of the present invention can be variously modified and modified without departing from the description of the claims.

In the above-described embodiment, the example in which the optical signal detection device is an inverted microscope is shown, but the optical signal detection device is not limited to the inverted microscope. Since the gel member has no fluidity, it can be observed diagonally or from the side. Therefore, as shown in FIG. 20, for example, when observing the temporal region of a relatively large animal S1 such as a mouse or a marmoset alive, an objective lens tilted toward the temporal region of the animal S1 is used. The optical signal detection device may perform imaging with the gel member 310 sandwiched between the 301 and the temporal region of the animal S1. In this case, the cover glass C1 is embedded in the temporal region of the animal S1 by an operation performed in advance, and the space between the cover glass C1 and the objective lens 301 may be filled with the gel member 310.

In the above-described embodiment, an example is shown in which the gel member is first attached to the objective lens, and then the space between the container and the objective lens is filled with the gel member by bringing the objective lens closer to the container. , The space between the container and the objective lens may be filled, and the procedure for attaching the gel member is not particularly limited. For example, as shown in FIGS. 21 and 22, observation may be performed using a microplate C2 having a large number of wells to which a gel member 410 is attached to the back surface. The gel member 410 preferably has a quarter consistency of 44 or more and 111 or less, and is deformed according to the shape of the back surface of the microplate C2 so as to be in close contact with the back surface of the microplate C2 without any gap. .. In this case, by pressing the objective lens against the gel member 410 on the back surface of the microplate C2, the space between the objective lens and the microplate C2 can be filled with the gel member 410, so that the sample can be easily observed with a high numerical aperture. Can be done.

In the above-described embodiment, an existing immersion objective lens designed for an immersion medium such as silicone oil or water is used, and a gel member is used instead of the immersion medium. An objective lens newly designed according to the gel member may be used. As a result, the objective lens can be newly designed according to the assumed refractive index of the gel member, so that the degree of freedom in selecting the gel member is increased.

1,100,200 Microscope device 3,107,206,301 Objective lens 6 1/4 cone 7 Test object 10-12, 310,410 Gel member C Container C1 Cover glass C2 Microplate L Distance P1 to P6 Microscope image S Sample S1 animal

Claims (24)

With the objective lens
A holding member that holds the sample between the objective lens and the sample,
A gel member that fills the space between the objective lens and the holding member.
The gel member is an optical signal detection device having a 1/4 consistency which is measured based on a consistency test using a 1/4 cone of JIS K 2220 and shows a value of 44 or more and 111 or less. In the optical signal detection device according to claim 1,
An optical signal detection device characterized in that the contact area of the gel member with the objective lens is larger than the contact area of the gel member with the holding member. With the objective lens
A holding member that holds the sample between the objective lens and the sample,
A gel member that fills the space between the objective lens and the holding member.
An optical signal detection device characterized in that the contact area of the gel member with the objective lens is larger than the contact area of the gel member with the holding member. In the optical signal detection device according to any one of claims 1 to 3, further
A revolver equipped with the objective lens is provided.
An optical signal detection device characterized in that when the revolver rotates, the gel member adsorbed on the objective lens moves together with the objective lens to the outside of the optical path of the objective lens. The optical signal detection device according to any one of claims 1 to 4.
The gel member is an optical signal detection device characterized in that it is detachably attached to the objective lens. The optical signal detection device according to any one of claims 1 to 5.
The gel member is an optical signal detection device having a tapered shape in which the cross-sectional area decreases from the objective lens toward the holding member. In the optical signal detection device according to claim 6,
The gel member is an optical signal detection device having a truncated cone shape. The optical signal detection device according to any one of claims 1 to 6.
The gel member is an optical signal detection device having a convex surface formed by a curved surface on the holding member side. The optical signal detection device according to any one of claims 1 to 8.
An optical signal detection device characterized in that the thickness of the gel member is 1.1 times or more and 1.5 times or less the working distance of the objective lens. The optical signal detection device according to any one of claims 1 to 9.
An optical signal detection device characterized in that the difference in refractive index between the gel member and the holding member is within ± 0.1. The optical signal detection device according to any one of claims 1 to 10.
An optical signal detection device characterized in that the difference in refractive index between the gel member and the sample or the medium covering the sample is within ± 0.1. A gel member attached to the tip of the objective lens.
The gel member is characterized by having a 1/4 consistency indicating a value of 44 or more and 111 or less, which is measured based on a consistency test using a 1/4 cone of JIS K 2220. In the gel member according to claim 12,
The gel member is a gel member that is detachably attached to the objective lens. In the gel member according to claim 12 or 13.
The gel member is characterized by having a tapered shape in which the cross-sectional area decreases in one direction. In the gel member according to claim 14,
The gel member is a gel member having a truncated cone shape. The gel member according to any one of claims 12 to 14.
The gel member is a gel member having a convex surface formed by a curved surface. The gel member according to any one of claims 13 to 16.
The gel member is characterized by having a refractive index within the range of 1.52 ± 0.1. The gel member according to any one of claims 13 to 17.
The gel member is characterized in that the thickness of the gel member is 1.1 times or more and 1.5 times or less the working distance of the objective lens. The gel member according to any one of claims 13 to 18.
A gel member characterized in that the contact area of the gel member with the objective lens is larger than the contact area of the gel member with a holding member for holding a sample. The space between the objective lens and the holding member that holds the sample has a 1/4 consistency that indicates a value of 44 or more and 111 or less, which is measured based on a consistency test using a 1/4 cone of JIS K 2220. Illuminating the sample with the gel member filled,
A method for detecting an optical signal, comprising detecting light from the sample incident via the objective lens with a photodetector. In the optical signal detection method according to claim 20, further
The distance in the optical axis direction of the objective lens between the objective lens and the sample is changed.
A method for detecting an optical signal, which comprises repeating the process of illuminating the sample and detecting the light from the sample with a photodetector every time the distance is changed. The optical signal detection device according to any one of claims 1 to 11.
The optical signal detection device, wherein the objective lens is an immersion type objective lens. In the gel member according to any one of claims 12 to 19.
The objective lens is a gel member characterized by being an immersion type objective lens. 20. In the optical signal detection method according to claim 21,
A method for detecting an optical signal, wherein the objective lens is an immersion type objective lens.

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