Method of verifying operation of fluorescence observation device, and pseudofluorescent phantom

Abstract

This method of operating a fluorescence observation device comprises: preparing a pseudofluorescent phantom 11 that is constituted of an absorbent material for absorbing a liquid 12, and contains a fluorescent substance made to generate fluorescence by excitation light Lp; irradiating the pseudofluorescent phantom 11 with the excitation light Lp before absorption of the liquid 12; detecting a first fluorescence Lf1 generated by the pseudofluorescent phantom 11; irradiating the pseudofluorescent phantom with the excitation light Lp after absorption of the liquid 12; detecting a second fluorescence Lf2 generated by the pseudofluorescent phantom 11; and verifying whether the fluorescence observation device 1 operates properly on the basis of a comparison of a luminance value of the first fluorescence Lf1 and a luminance value of the second fluorescence Lf2.

Description

Operation verification method for fluorescence observation device and pseudo-fluorescent phantom

[0001] The present disclosure relates to an operation verification method for a fluorescence observation device and a pseudo-fluorescent phantom.

[0002] Fluorescence observation devices are known that inspect biological samples by irradiating excitation light onto a biological sample containing a reagent (fluorescent probe) and observing the emitted fluorescence. For example, the fluorescence observation devices described in Patent Documents 1 and 2 include a tray, a light source, a detection unit, and an image generation unit. In these fluorescence observation devices, a specimen (sample) stained with a fluorescent probe is placed on the tray, and the tray is set at an inspection position within the housing. Next, excitation light is irradiated onto the specimen from the light source, and fluorescence generated in the specimen by irradiation with the excitation light is detected. The inspection of the specimen is performed by comparing the fluorescence brightness value after a predetermined time has elapsed with a predetermined threshold.

[0003] JP 2020-153966 A JP 2020-160012 A

[0004] When using a fluorescence observation device like the one described above, verification is performed to determine whether the device is operating normally, for example, before starting use. Ideally, operational verification would involve irradiating an actual sample with excitation light and measuring changes in the fluorescence state over time. However, this requires complex procedures such as preparing reagents (fluorescent probes) and controlling their temperature, making it unsuitable for on-site operation. While using a hard sample such as a fluorescent screen could be considered, changing the fluorescence state over time would require the addition of some kind of driving mechanism, which could necessitate securing a power source and space around the device.

[0005] The present disclosure has been made to solve the above-described problems, and has an object to provide an operation verification method for a fluorescence observation device and a pseudo-fluorescent phantom that can easily verify the operation of a fluorescence observation device.

[0006] The gist of the present disclosure is as follows.

[0007] [1] A method for verifying the operation of a fluorescence observation device that is the target of performance verification, the method comprising the steps of: a preparation step of preparing a pseudo-fluorescent phantom that is made of an absorbent material that absorbs liquid and that contains a fluorescent substance that generates fluorescence in response to excitation light; a first detection step of irradiating the pseudo-fluorescent phantom with excitation light before the liquid is absorbed and detecting first fluorescence generated in the pseudo-fluorescent phantom; a second detection step of irradiating the pseudo-fluorescent phantom with excitation light after the liquid is absorbed and detecting second fluorescence generated in the pseudo-fluorescent phantom; and a verification step of verifying whether the fluorescence observation device operates normally based on a comparison of a luminance value of the first fluorescence and a luminance value of the second fluorescence.

[0008] This operational verification method for a fluorescence observation device uses a pseudo-fluorescent phantom instead of an actual sample and verifies whether the fluorescence observation device operates normally based on the temporal change in fluorescence intensity values ​​of the pseudo-fluorescent phantom before and after absorbing liquid. The pseudo-fluorescent phantom is composed of an absorptive material that absorbs liquid and contains a fluorescent substance that emits fluorescence in response to excitation light. By absorbing liquid, the pseudo-fluorescent phantom generates a new fluorescent state while maintaining the fluorescence wavelength to be detected, allowing it to behave similarly to when observing an actual sample. Therefore, this operational verification method for a fluorescence observation device does not require the preparation or temperature control of reagents (fluorescent probes), and does not require additional driving mechanisms compared to using hard samples such as fluorescent screens, making it easy to verify the operation of the fluorescence observation device.

[0009] [2] The method for verifying the operation of a fluorescence observation device according to [1], wherein the preparation step prepares a pseudo-fluorescent phantom made of an absorbent material that expands upon absorbing the liquid. In this case, the pseudo-fluorescent phantom generates a new fluorescence region by absorbing the liquid while maintaining the fluorescence wavelength to be detected. Therefore, the behavior of the pseudo-fluorescent phantom becomes closer to the behavior of an actual sample, further improving the accuracy of the operation verification of the fluorescence observation device.

[0010] [3] The method for verifying the operation of a fluorescence observation device according to [1] or [2], wherein water is used as the liquid to be absorbed into the pseudo-fluorescent phantom in the second detection step. In this case, by using water, which is easy to obtain and handle, the operation of the fluorescence observation device can be verified more easily.

[0011] [4] The method for verifying the operation of a fluorescence observation device according to [1] or [2], wherein the second detection step uses a liquid containing a fluorescent substance as the liquid to be absorbed by the pseudo-fluorescent phantom. In this case, the fluorescence brightness value of the pseudo-fluorescent phantom increases over time due to absorption of the liquid. Therefore, the behavior of the pseudo-fluorescent phantom becomes closer to the behavior of an actual sample, further improving the accuracy of the operation verification of the fluorescence observation device.

[0012] [5] A pseudo-fluorescent phantom used for verifying the operation of a fluorescence observation device, which is made of an absorbent material that absorbs liquid and contains a fluorescent substance that generates fluorescence when exposed to excitation light.

[0013] This pseudo-fluorescent phantom absorbs liquid and generates a new fluorescent state while maintaining the fluorescent wavelength to be detected, allowing it to behave in the same way as when observing an actual sample. Therefore, using this pseudo-fluorescent phantom eliminates the need for reagent (fluorescent probe) preparation and temperature control, and, unlike when using a hard sample such as a fluorescent screen, no additional driving mechanism is required, making it easy to verify the operation of a fluorescence observation device.

[0014] [6] The pseudo-fluorescent phantom according to [5], wherein the absorbing material is made of a material that expands upon absorbing the liquid. In this case, the pseudo-fluorescent phantom generates a new fluorescence region by absorbing the liquid while maintaining the fluorescence wavelength to be detected. Therefore, the behavior of the pseudo-fluorescent phantom is closer to the behavior of an actual sample, further improving the accuracy of operational verification of a fluorescence observation device.

[0015] According to the present disclosure, the operation of a fluorescence observation device can be easily verified.

[0016] 1 is a block diagram illustrating an example of a fluorescence observation device. (a) is a diagram illustrating a pseudo-fluorescent phantom before liquid absorption, and (b) is a diagram illustrating the pseudo-fluorescent phantom before liquid absorption. (a) to (c) are diagrams schematically illustrating changes over time in the fluorescence state of a pseudo-fluorescent phantom when water is absorbed. (a) to (d) are diagrams schematically illustrating changes over time in the fluorescence state of a pseudo-fluorescent phantom when a liquid containing a fluorescent substance is absorbed. (b) is a flowchart illustrating an example of a method for verifying the operation of a fluorescence observation device. (a) is an example of a visible image of a pseudo-fluorescent phantom immediately after liquid absorption, and (b) is an example of a visible image of a pseudo-fluorescent phantom after liquid absorption. (a) is an example of a fluorescence image of a pseudo-fluorescent phantom immediately after liquid absorption, (b) is an example of a fluorescence image of a pseudo-fluorescent phantom after liquid absorption, and (c) is an example of a difference image before and after liquid absorption. (a) is an example of a fluorescent image of a pseudo-fluorescent phantom immediately after absorbing liquid, (b) is an example of a fluorescent image of a pseudo-fluorescent phantom immediately after absorbing liquid, and (c) is an example of a fluorescent image of a pseudo-fluorescent phantom a predetermined time after absorbing liquid.

[0017] Hereinafter, preferred embodiments of a method for verifying operation of a fluorescence observation device and a pseudo-fluorescent phantom according to one aspect of the present disclosure will be described in detail with reference to the drawings.

[0018] FIG. 1 is a block diagram showing an example of a fluorescence observation device. The fluorescence observation device 1 shown in FIG. 1 is configured as a device for performing fluorescence observation of a biological sample S immersed in a liquid reagent (fluorescent probe). In this embodiment, an example application of the fluorescence observation device 1 is a margin inspection of tumor tissue excised from a patient by surgery. In the margin inspection, the biological sample S containing tumor tissue is stained with a fluorescent probe. The behavior of the fluorescence intensity over time differs between tumor tissue and normal tissue. Therefore, by measuring the fluorescence brightness of the biological sample S after a predetermined time has elapsed since staining with the fluorescent probe, it is possible to determine whether or not tumor tissue remains in the biological sample S.

[0019] A fluorescent probe is a substance whose molecular structure changes upon reaction with a specific substance, emitting strong fluorescence. An example of a fluorescent probe used in stump testing is gGlu-HMRG. gGlu-HMRG is a liquid fluorescent substance. Before reaction, gGlu-HMRG is colorless and transparent, and water-soluble. After reacting with an enzyme derived from tumor tissue, gGlu-HMRG becomes colored and is visualized. It also changes from water-soluble to hydrophobic, and exhibits the property of permeating cell membranes and remaining within the cells. Therefore, the use of gGlu-HMRG enables accurate detection of even minute tumor tissue, for example, a few millimeters in size.

[0020] 1, the fluorescence observation device 1 includes a tray 2, a light source unit 3, a detection unit 4, and an image generation unit 5. These components are arranged inside a housing (not shown) that blocks external light. The fluorescence observation device 1 is connected to a monitor 6 via a wired or wireless connection so that information can be communicated therewith. The monitor 6 can be configured, for example, by a personal computer or a smart device (such as a smartphone or tablet terminal).

[0021] The tray 2 is a member for setting the biological sample S in the fluorescence observation device 1. The tray 2 is formed, for example, in the shape of a rectangular plate in a plan view. From the viewpoint of ensuring sensitivity of the fluorescence observation of the biological sample S, the tray 2 preferably has a dark color such as black. By placing the tray 2 with the biological sample S placed thereon inside the housing, the biological sample S can be placed at the examination position T of the fluorescence observation device 1.

[0022] The light source unit 3 includes an illumination light source 7 and an excitation light source 8. The illumination light source 7 and the excitation light source 8 are disposed, for example, in the housing, above the position where the tray 2 is disposed. The illumination light source 7 is a white light source constituted, for example, by a lamp. The illumination light source 7 irradiates the biological sample S on the tray 2 from above with illumination light Ls. The excitation light source 8 is constituted, for example, by an LED. The excitation light source 8 irradiates the biological sample S on the tray 2 from above with excitation light Lp.

[0023] The detection unit 4 is configured to include an imaging element such as a CCD image sensor or a CMOS image sensor. The detection unit 4 is disposed, for example, above the position of the tray 2 inside the housing. The detection surface of the detection unit 4 is disposed facing downward so as to face the tray 2. The detection unit 4 may include an optical filter 9, as necessary, that transmits fluorescence of a wavelength generated in the biological sample S. The detection unit 4 outputs, to the image generation unit 5, a detection signal G1 that indicates the image capture result of the biological sample S using the illumination light Ls, and a detection signal G2 that indicates the image capture result of the fluorescence generated in the biological sample S when irradiated with the excitation light Lp.

[0024] The image generating unit 5 is configured by, for example, a microcomputer equipped with a processor and a memory or an FPGA (field-programmable gate array). The image generating unit 5 generates a fluorescent image of the biological sample S based on the detection signal from the detecting unit 4. In this embodiment, the image generating unit 5 generates a visible image based on a detection signal G1 indicating the imaging result of the biological sample S using illumination light, and generates a fluorescent image based on a detection signal G2 indicating the imaging result of fluorescence generated in the biological sample S. The image generating unit 5 generates a superimposed image by superimposing the visible image and the fluorescent image, and outputs the superimposed image to the monitor 6.

[0025] The operation verification method for a fluorescence observation device according to the present disclosure is applied to the above-described fluorescence observation device 1. Operation verification is a task performed to verify whether the fluorescence observation device 1 operates correctly (i.e., whether it is capable of correctly detecting changes over time in the luminance values ​​of fluorescence generated in the biological sample S), and is performed appropriately at timings such as the first fluorescence observation, each fluorescence observation, and maintenance.

[0026] In this embodiment, a pseudo-fluorescent phantom 11 as shown in FIGS. 2( a) and 2(b) is used in place of an actual biological sample S to verify operation. The pseudo-fluorescent phantom 11 is made of an absorbing material that absorbs a liquid 12 and contains a fluorescent substance that generates fluorescence when exposed to excitation light Lp. Examples of the absorbing material that makes up the pseudo-fluorescent phantom 11 include polyacrylate-based water-absorbent polymers. The absorbing material may also be a biodegradable polyacrylate-based water-absorbent polymer.

[0027] The fluorescent substance contained in the pseudo fluorescent phantom 11 may be a fluorescent probe used for observing an actual biological sample S. In this embodiment, gGlu-HMRG is exemplified as the fluorescent probe used for examining the margin of the biological sample S, and the pseudo fluorescent phantom 11 also contains gGlu-HMRG as a fluorescent substance.

[0028] The liquid 12 to be absorbed into the pseudo-fluorescent phantom 11 is not particularly limited as long as it can be absorbed by an absorbent material, does not inhibit the generation of fluorescence by the excitation light Lp, and does not decompose or dissolve the absorbent material. Here, water or a liquid containing a fluorescent substance is exemplified as the liquid 12. The water may be pure water or tap water. When a liquid containing a fluorescent substance is used, the fluorescent substance may be the same as or different from the fluorescent substance contained in the pseudo-fluorescent phantom 11. In the latter case, the liquid 12 may be, for example, an aqueous solution of a vitamin supplement.

[0029] The pseudo-fluorescent phantom 11 gradually expands as it absorbs the liquid 12. There are no particular restrictions on the volume ratio of the pseudo-fluorescent phantom 11 before and after absorbing the liquid, but if the volume of the pseudo-fluorescent phantom 11 before absorbing the liquid is taken as 1, the maximum volume of the pseudo-fluorescent phantom 11 after absorbing the liquid may be approximately 100 to 200. The time from the start of absorbing the liquid until the volume of the pseudo-fluorescent phantom 11 reaches its maximum volume is preferably shorter than the time it takes to observe the actual biological sample S in the fluorescence observation device 1. As an example, the time from the start of absorbing the liquid until the volume of the pseudo-fluorescent phantom 11 reaches its maximum volume is preferably within 15 to 20 minutes.

[0030] Note that "before absorbing liquid" here includes not only the state before the liquid 12 is absorbed into the pseudo-fluorescent phantom 11, but also the state immediately after the liquid 12 is absorbed into the pseudo-fluorescent phantom 11. "Immediately after" essentially refers to the state before the absorbing of the liquid 12 into the pseudo-fluorescent phantom 11 begins. For example, "immediately after" corresponds to the state within 15 seconds after the liquid 12 is dripped onto the pseudo-fluorescent phantom 11. As will be described later, when calculating the difference between the state before absorbing liquid and the state after absorbing liquid, if the position of the pseudo-fluorescent phantom 11 is shifted due to the dripping of the liquid 12, the accuracy of the difference image may be reduced. Therefore, by including the state immediately after absorbing liquid in "before absorbing liquid," the accuracy of the difference image can be ensured.

[0031] 2(a) and 2(b), the shape of the pseudo-fluorescent phantom 11 is spherical. The shape of the pseudo-fluorescent phantom 11 is not limited to a sphere, and may be other shapes such as an ellipsoid, a polyhedron, a cube, a rectangular parallelepiped, a cylinder, or a cone. To improve the liquid absorption efficiency, the surface of the pseudo-fluorescent phantom 11 may be provided with protrusions, recesses, grooves, notches, or the like. For the same reason, at least a portion of the surface of the pseudo-fluorescent phantom 11 may be roughened.

[0032] Next, the function of the pseudo fluorescent phantom 11 will be described. Figures 3(a) to 3(c) are diagrams schematically showing the change over time in the fluorescent state of the pseudo fluorescent phantom 11 when it absorbs water. In these diagrams, the horizontal axis represents the spatial size of the pseudo fluorescent phantom 11, and the vertical axis represents the fluorescent intensity. In the initial state before water absorption, the fluorescent light (first fluorescent light Lf1) emitted from the pseudo fluorescent phantom 11 has a spatial size W1 corresponding to the diameter of the pseudo fluorescent phantom 11, and a fluorescent light intensity A, as shown in Figure 3(a).

[0033] After absorbing water, the total amount of fluorescence from the pseudo fluorescent phantom 11 remains unchanged, while the volume of the pseudo fluorescent phantom 11 increases. Therefore, as shown in FIG. 3( b), the fluorescence (second fluorescence Lf2) generated from the pseudo fluorescent phantom 11 has a spatial size W2 that is larger than the initial spatial size W1, and the overall fluorescence intensity B is smaller than the initial fluorescence intensity A. Therefore, when the difference between the state before and after absorbing water is taken, as shown in FIG. 3( c), the fluorescence in the region corresponding to the initial spatial size W1 disappears, but instead a new fluorescent region Lfn having a fluorescence intensity B smaller than the initial fluorescence intensity A is generated in the region outside the initial spatial size W1.

[0034] 4( a ) to 4 ( d ) are diagrams schematically illustrating the change over time in the fluorescence state of the quasi-fluorescent phantom 11 when it is made to absorb a liquid containing a fluorescent substance. In these diagrams, the horizontal axis also represents the spatial size of the quasi-fluorescent phantom 11, and the vertical axis represents the fluorescence intensity. In the initial state before the quasi-fluorescent phantom 11 absorbs the liquid containing a fluorescent substance, the fluorescence (first fluorescence Lf1) generated from the quasi-fluorescent phantom 11 has a spatial size W1 corresponding to the diameter of the quasi-fluorescent phantom 11 and a fluorescence intensity A, as shown in FIG. 4( a ). As shown in FIG. 4( b ), immediately after the quasi-fluorescent phantom 11 absorbs the liquid containing a fluorescent substance, the liquid with fluorescence intensity A generates a fluorescence intensity A around the quasi-fluorescent phantom 11.

[0035] After the quasi-fluorescent phantom 11 absorbs the liquid containing the fluorescent substance, the total amount of fluorescence from the quasi-fluorescent phantom 11 increases, and the volume of the quasi-fluorescent phantom 11 also increases. Therefore, as shown in FIG. 4( c), the fluorescence (second fluorescence Lf2) generated from the quasi-fluorescent phantom 11 assumes a spatial size W2 larger than the initial spatial size W1, and the overall fluorescence intensity also changes to a fluorescence intensity C larger than the initial fluorescence intensity A. Therefore, when the difference between the state before and after the absorption is calculated, as shown in FIG. 4( d), a fluorescence change region Lfm having a fluorescence intensity (C-A) is generated in the region corresponding to the initial spatial size W1, and a new fluorescence region Lfn having a fluorescence intensity (C-A) is generated in the region outside the initial spatial size W1. Note that, for convenience of explanation, in FIGS. 4( a) to 4( d), the fluorescence intensity of the fluorescent substance contained in the liquid is set to be the same as the fluorescence intensity of the quasi-fluorescent phantom; however, the fluorescence intensity of the fluorescent substance contained in the liquid may differ from the fluorescence intensity of the quasi-fluorescent phantom.

[0036] 5 is a flowchart showing an example of a method for verifying the operation of a fluorescence observation device. As shown in the figure, the method for verifying the operation of a fluorescence observation device according to this embodiment sequentially performs a preparation step S01, a first detection step S02, a second detection step S03, and a verification step S04 in the fluorescence observation device 1 that is the target of the operation verification. The preparation step S01 is a step for preparing the above-mentioned pseudo-fluorescence phantom 11. In the preparation step S01, the pseudo-fluorescence phantom 11 and a liquid 12 (here, water or a liquid containing a fluorescent substance) to be absorbed into the pseudo-fluorescence phantom 11 are also prepared.

[0037] The first detection step S02 is a step of irradiating the quasi-fluorescent phantom 11 with excitation light Lp before (including immediately after) absorbing the liquid 12, and detecting the first fluorescence Lf1 generated in the quasi-fluorescent phantom 11. The first fluorescence Lf1 is fluorescence corresponding to the fluorescence state illustrated in FIG. 3( a) or FIG. 4( a). In the first detection step S02, the tray 2 on which the quasi-fluorescent phantom 11 is placed is installed in the housing, and the quasi-fluorescent phantom 11 is placed at the examination position T of the fluorescence observation device 1. In this state, the quasi-fluorescent phantom 11 is irradiated with excitation light Lp from the light source unit 3. The first fluorescence Lf1 generated in the quasi-fluorescent phantom 11 is then detected by the detection unit 4, and a first fluorescence image based on the first fluorescence Lf1 is generated by the image generation unit 5.

[0038] The second detection step S03 is a step in which excitation light Lp is irradiated onto the quasi-fluorescent phantom 11 after the liquid 12 has been absorbed, and second fluorescence Lf2 generated in the quasi-fluorescent phantom 11 is detected. The second fluorescence Lf2 is fluorescence corresponding to the fluorescent state exemplified in FIG. 3( b) or FIG. 4( c). In the second detection step S03, the tray 2 on which the quasi-fluorescent phantom 11 is placed is temporarily removed from the housing, and the quasi-fluorescent phantom 11 is allowed to absorb the liquid 12 for a predetermined period of time. This causes the quasi-fluorescent phantom 11 to expand, generating a new fluorescent state different from its initial state.

[0039] After the liquid 12 has been absorbed, the tray 2 on which the pseudo-fluorescent phantom 11 is placed is placed back into the housing, and the pseudo-fluorescent phantom 11 is placed at the examination position T of the fluorescence observation device 1. In this state, the excitation light Lp from the light source unit 3 is irradiated onto the pseudo-fluorescent phantom 11. The second fluorescence Lf2 generated by the pseudo-fluorescent phantom 11 is then detected by the detection unit 4, and a second fluorescence image based on the second fluorescence Lf2 is generated by the image generation unit 5.

[0040] The verification step S04 is a step of verifying whether or not the fluorescence observation device 1 is operating normally based on a comparison between the luminance value of the first fluorescence Lf1 and the luminance value of the second fluorescence Lf2. In this embodiment, the image generation unit 5 generates a difference image between the first fluorescence image acquired in the first detection step S02 and the second fluorescence image acquired in the second detection step S03.

[0041] In the verification step S04, for example, the number of pixels in the difference image that exhibit brightness values ​​exceeding a predetermined threshold is counted, and if the count number is equal to or greater than the predetermined number, it is determined that the operation of the fluorescence observation device 1 is normal, and if the count number is less than the predetermined number, it is determined that the operation of the fluorescence observation device 1 is abnormal. The threshold value set for the brightness value of the difference image may be the same as the threshold value used to determine the presence or absence of remaining tumor tissue in the actual biological sample S, or any value may be used depending on the type of fluorescent material contained in the pseudo fluorescent phantom 11. The verification step S04 may be performed visually based on the difference image displayed on the monitor 6. Alternatively, the fluorescence observation device 1 may be configured to include a verification unit that performs the verification step S04 based on the difference image received from the image generation unit 5.

[0042] As described above, this method for verifying the operation of a fluorescence observation device uses a pseudo-fluorescent phantom 11 instead of an actual biological sample S, and verifies whether the fluorescence observation device 1 operates normally based on the change over time in the fluorescence luminance value of the pseudo-fluorescent phantom 11 before and after absorbing liquid. The pseudo-fluorescent phantom 11 generates a new fluorescence state by absorbing the liquid 12 while maintaining the fluorescence wavelength to be detected, and therefore can behave in a manner similar to that when observing an actual biological sample S. Therefore, this method for verifying the operation of a fluorescence observation device does not require the preparation or temperature control of a reagent (fluorescent probe), and does not require an additional driving mechanism as compared to when using a hard sample such as a fluorescent screen, making it possible to easily verify the operation of the fluorescence observation device 1.

[0043] In this embodiment, in preparation step S01, a pseudo-fluorescent phantom 11 is prepared, which is made of an absorbent material that expands upon absorbing the liquid 12. In this case, the pseudo-fluorescent phantom 11 generates a new fluorescence region (new fluorescence region Lfn) while maintaining the fluorescence wavelength to be detected by absorbing the liquid 12. Therefore, the behavior of the pseudo-fluorescent phantom 11 becomes even closer to the behavior of an actual biological sample S, and the accuracy of operational verification of the fluorescence observation device 1 can be further improved.

[0044] This embodiment includes an aspect in which water is used as the liquid 12 to be absorbed into the pseudo-fluorescent phantom 11 in the second detection step S03. In this case, by using water, which is easy to obtain and handle, the operational verification of the fluorescence observation device 1 can be carried out even more easily.

[0045] This embodiment includes an aspect in which, in the second detection step S03, a liquid containing a fluorescent substance is used as the liquid 12 to be absorbed by the pseudo-fluorescent phantom 11. In this case, the luminance value of the fluorescence of the pseudo-fluorescent phantom 11 increases over time due to absorption by the liquid 12. Therefore, the behavior of the pseudo-fluorescent phantom 11 becomes even closer to the behavior of an actual biological sample S, and the accuracy of operational verification of the fluorescence observation device 1 can be further improved.

[0046] The pseudo-fluorescent phantom 11 used to verify the operation of the fluorescence observation device 1 is made of an absorptive material that absorbs the liquid 12 and contains a fluorescent substance that generates fluorescence in response to excitation light Lp. The pseudo-fluorescent phantom 11 generates a new fluorescent state by absorbing the liquid 12 while maintaining the fluorescence wavelength to be detected, and therefore can behave in a manner similar to that when observing an actual biological sample S. Therefore, use of the pseudo-fluorescent phantom 11 eliminates the need for preparation and temperature control of reagents (fluorescent probes), and also eliminates the need for an additional driving mechanism compared to when using a hard sample such as a fluorescent screen, making it possible to easily verify the operation of the fluorescence observation device 1.

[0047] In this embodiment, the absorbing material of the pseudo-fluorescent phantom 11 is made of a material that expands upon absorbing liquid. In this case, the pseudo-fluorescent phantom 11 generates a new fluorescence region (new fluorescence region Lfn) while maintaining the fluorescence wavelength to be detected by absorbing the liquid 12. Therefore, the behavior of the pseudo-fluorescent phantom 11 becomes even closer to the behavior of an actual biological sample S, and the accuracy of operational verification of the fluorescence observation device 1 can be further improved.

[0048] Examples of the present disclosure are described below. Here, multiple pseudo-fluorescent phantoms containing a polyacrylate-based water-absorbent polymer as the absorbing material and gGlu-HMRG as the fluorescent substance were placed on a tray, and actual images of the pseudo-fluorescent phantoms before and after absorbing the liquid were acquired to confirm their behavior.

[0049] FIG. 6( a) shows an example of a visible image of a pseudo-fluorescent phantom immediately after absorbing liquid. In the example of FIG. 6( a), a pseudo-fluorescent phantom was placed at each of the four corners of a tray, and a visible image was acquired by irradiating the pseudo-fluorescent phantom with illumination light immediately after absorbing liquid. FIG. 6( b) shows an example of a visible image of the pseudo-fluorescent phantom a predetermined time after absorbing liquid. In the example of FIG. 6( b), the pseudo-fluorescent phantom in the state of FIG. 6( a) was immersed in water (tap water) for 30 minutes to absorb liquid, and then irradiated with illumination light to acquire a visible image. The results of FIGS. 6( a) and 6( b) confirm that the volume of the pseudo-fluorescent phantom increased by one size due to absorbing liquid.

[0050] FIG. 7( a) is an example of a fluorescence image of a pseudo-fluorescent phantom immediately after liquid absorption. The example of FIG. 7( a) was obtained by irradiating excitation light onto the pseudo-fluorescent phantom in the state shown in FIG. 6( a). This fluorescence image shows that each of the pseudo-fluorescent phantoms at the four corners of the tray emits fluorescence with a constant brightness value. FIG. 7( b) is an example of a fluorescence image of a pseudo-fluorescent phantom after liquid absorption. The example of FIG. 7( b) was obtained by irradiating excitation light onto the pseudo-fluorescent phantom in the state shown in FIG. 6( b). This fluorescence image shows that the fluorescence brightness values ​​of each pseudo-fluorescent phantom are smaller than those in the state shown in FIG. 6( a), while the fluorescent region increases in size as the volume of the pseudo-fluorescent phantom increases.

[0051] Fig. 7(c) shows an example of a difference image before and after liquid absorption. The example in Fig. 7(c) is an image obtained by subtracting the fluorescence image in Fig. 7(a) from the fluorescence image in Fig. 7(b). This difference image confirmed that when the pseudo-fluorescent phantom was made to absorb water, the initial fluorescent region disappeared, but instead a new fluorescent region was generated around it.

[0052] Figures 8(a) to 8(c) show an example using a liquid containing a fluorescent substance. Here, green pseudo-fluorescent phantoms were placed in two locations on the tray (the bottom rows of Figures 8(a) to 8(c)), and red pseudo-fluorescent phantoms were placed in two other locations on the tray (the bottom rows of Figures 8(a) to 8(c)). A detector with sensitivity near a wavelength of 520 nm was used to capture the fluorescent images.

[0053] FIG. 8( a) shows an example of a fluorescence image of a pseudo-fluorescent phantom immediately after liquid absorption. This fluorescence image reveals that, in the initial state immediately after liquid absorption, no fluorescence is emitted from the red pseudo-fluorescent phantom, but fluorescence is emitted from the green pseudo-fluorescent phantom. FIG. 8( b) shows an example of a fluorescence image of a pseudo-fluorescent phantom immediately after liquid absorption. Water (tap water) was dripped onto the two pseudo-fluorescent phantoms on the left side of the drawing, and a liquid containing a fluorescent substance (aqueous vitamin supplement solution) was dripped onto the two pseudo-fluorescent phantoms on the right side of the drawing. This fluorescence image shows that there is no change in either the red or green pseudo-fluorescent phantom immediately after water is dripped onto them, but fluorescence from the liquid containing a fluorescent substance can be confirmed immediately after dripping onto them.

[0054] Figure 8(c) shows an example of a fluorescence image of the pseudo-fluorescent phantom after a predetermined time had elapsed since the liquid absorption. The liquid absorption time was 30 minutes, as in Figures 6(b) and 7(b). This fluorescence image confirmed that a fluorescence change region was generated in the center of the green pseudo-fluorescent phantom, where the fluorescence intensity value changed from the initial state, and that a new fluorescent region was generated around it. Furthermore, it was confirmed that the red pseudo-fluorescent phantom, which did not emit fluorescence in the initial state, also became a new fluorescent region as a whole due to the absorption of the liquid containing the fluorescent substance.

[0055] 1...fluorescence observation device, 11...pseudo-fluorescence phantom, 12...liquid, Lp...excitation light, Lf1...first fluorescence, Lf2...second fluorescence.

Claims

1. A method for verifying the operation of a fluorescence observation device that is the subject of operational verification, which sequentially carries out the following steps in a fluorescence observation device: a preparation step of preparing a pseudo-fluorescent phantom that is made of an absorbent material that absorbs liquid and contains a fluorescent substance that emits fluorescence in response to excitation light; a first detection step of irradiating the pseudo-fluorescent phantom with excitation light before the liquid is absorbed and detecting first fluorescence generated in the pseudo-fluorescent phantom; a second detection step of irradiating the pseudo-fluorescent phantom with excitation light after the liquid is absorbed and detecting second fluorescence generated in the pseudo-fluorescent phantom; and a verification step of verifying whether the fluorescence observation device is operating normally based on a comparison of the luminance value of the first fluorescence and the luminance value of the second fluorescence.

2. A method for verifying the operation of a fluorescence observation device according to claim 1, wherein the preparation step includes preparing a pseudo-fluorescence phantom made of an absorbent material that expands when it absorbs the liquid.

3. A method for verifying the operation of a fluorescence observation device according to claim 1 or 2, wherein in the second detection step, water is used as the liquid to be absorbed into the pseudo-fluorescent phantom.

4. A method for verifying the operation of a fluorescence observation device according to claim 1 or 2, wherein in the second detection step, a liquid containing a fluorescent substance is used as the liquid to be absorbed into the pseudo-fluorescent phantom.

5. A pseudo-fluorescent phantom used to verify the operation of a fluorescence observation device, which is made of an absorbent material that absorbs liquid and contains a fluorescent substance that emits fluorescence when exposed to excitation light.

6. A pseudo-fluorescent phantom according to claim 5, wherein said absorbing material is made of a material that expands upon absorbing said liquid.

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