Photoacoustic wave generation sheet, photoacoustic measurement method, and optical hardness measuring method

Abstract

To provide a photoacoustic wave generation sheet that efficiently generates a photoacoustic wave for sample excitation without using high-intensity pulse light or special petri dishes.SOLUTION: A photoacoustic wave generation sheet 71 is used in photoacoustic measurement of a sample 2 placed in contact with a first surface 3a of a sample contact body 3. The sheet 71 is placed in contact with a second surface 3b located on an opposite side of the first surface 3a. The sheet 71 comprises a light reflector 73, light absorbers 74, and a photoacoustic wave generator 72. The light reflector 73 reflects measurement light L2. The light absorbers 74 are present at positions shifted in a surface direction of the sheet 71 from the light reflector 73, in order to absorb exciting pulse light L1 and convert it to heat energy. The light-permeable photoacoustic wave generator 72 is placed in contact with the light absorbers 74, and its thermal expansion coefficient is larger than that of the light absorbers 74.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a photoacoustic wave generating sheet, a photoacoustic measuring method, and an optical hardness measuring method. [Background technology]

[0002] Generally, mechanical properties can be determined by applying an external force to a sample and measuring the displacement. However, when the sample is a small single cell, it is difficult to measure the displacement because the cell is very weakly resistant to external forces and easily destroyed. Conventional methods for measuring the mechanical properties of cells include, for example, a method using an atomic force microscope. However, since it is necessary to bring the probe close to the cell, there is a concern that the cell may be contaminated or damaged, and this is not an effective method for measuring mechanical properties. In addition, the method using an atomic force microscope is completely different from cell observation using a normal optical microscope, and there are many barriers to its use by researchers in the field of biology.

[0003] In order to overcome these drawbacks, it is essential to establish a method for measuring the mechanical properties of cells without contact while sealing the cell culture dish and maintaining the culture environment. Under these circumstances, the inventors of the present application have previously proposed a method for measuring the hardness of cells by vibrating a plastic cell culture dish and optically measuring the displacement (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2022-163812 A Summary of the Invention [Problem to be solved by the invention]

[0005] Here, according to an experiment conducted by the inventors of the present application, it has become clear that in order to realize optical hardness measurement, strong pulsed light of about 30 μJ / pulse is required for excitation. For excitation by pulsed light, it is necessary to use pulsed light with a pulse width of about 10 nanoseconds. When such pulsed light is irradiated onto a plastic petri dish made of polystyrene or the like, the temperature of the plastic petri dish rises instantaneously due to light absorption, and the volume expands, generating photoacoustic waves. However, typical plastic petri dishes often used in cell culture absorb only about 5% of the pulsed light. As a result, the efficiency of converting pulsed light into photoacoustic waves is 5% or less.

[0006] In order to realize optical hardness measurement, the inventors of this application have been considering a method of irradiating cells with strong pulsed light without damaging them by adjusting the distribution of light. However, a large pulsed light generating unit is required to obtain strong pulsed light, and the entire device is inevitably large in scale. In addition, it is necessary to select a special petri dish suitable for measurement.

[0007] On the other hand, the choice of the petri dish to use for measurement depends on the researcher's preference and compatibility with the experiment. For this reason, it would be preferable to be able to measure hardness with many different types of petri dishes without being limited to a specific type.

[0008] The present invention has been made in view of the above problems, and an object of the present invention is to provide a photoacoustic wave generating sheet capable of efficiently generating photoacoustic waves for sample vibration without using high-intensity pulsed light or a special petri dish. Another object of the present invention is to provide a photoacoustic measurement method and an optical hardness measurement method that can perform desired measurements in a non-contact manner at a relatively low cost and easily by using the above-mentioned suitable photoacoustic wave generating sheet. [Means for solving the problem]

[0009] Means for solving the above problems are listed below in [1] to

[12] . [1] A photoacoustic wave generating sheet that is placed in contact with a second surface opposite to the first surface of a sample contact body for photoacoustic measurement of a sample placed in contact with the first surface, the photoacoustic wave generating sheet comprising: a light reflector that reflects measurement light; a light absorber that is at least at a position shifted from the light reflector in the planar direction of the sheet and absorbs excitation pulsed light and converts it into thermal energy; and a light-transmitting photoacoustic wave generator that is placed in contact with the light absorber and has a thermal expansion coefficient larger than that of the light absorber. [2] The photoacoustic wave generating sheet described in 1 above, characterized in that the photoacoustic wave generator is a transparent resin layer that covers the light reflector and the light absorber, and is formed with a larger area and uniform thickness than them. [3] The photoacoustic wave generating sheet according to item 2 above, wherein the light reflector is a metal layer, and the light absorber is a microparticle dispersion layer in which inorganic microparticles that absorb light are dispersed. [4] The photoacoustic wave generating sheet described in 3 above, characterized in that the photoacoustic wave generating sheet is a photoacoustic wave generating tape that is flexible overall and can be attached to and peeled off from the first surface of the sample contact body. [5] A photoacoustic wave generating sheet described in any one of items 1 to 4 above, further comprising a light-transmitting adhesive layer formed on one side of the sheet, and a protective layer formed in a peelable manner so as to cover the adhesive layer. [6] A photoacoustic wave generating sheet described in any one of items 1 to 4 above, characterized in that the light reflector is formed so as to occupy an area of ​​less than 50% of the area of ​​the photoacoustic wave generating sheet. [7] A photoacoustic wave generating sheet described in any one of claims 1 to 4, characterized in that a plurality of the light reflectors and a plurality of the light absorbers are formed, and the gaps between the plurality of the light reflectors and the gaps between the plurality of the light absorbers are both smaller than the size of the sample. [8] A photoacoustic wave generating sheet described in any one of items 1 to 4 above, characterized in that the size of the light absorber is larger than the wavelength of the photoacoustic wave generated, and the thicknesses of the light absorber and the photoacoustic wave generator are both equal to or greater than the wavelength of the photoacoustic wave generated. [9] The photoacoustic wave generating sheet described in any one of items 1 to 4 above, characterized in that the sample contact body is the bottom of a plastic petri dish, and the sample is a cell.

[10] The photoacoustic wave generating sheet described in any one of claims 1 to 4, characterized in that the sample contact body is a tip placement member provided at the tip of an endoscope, and the sample is in vivo tissue.

[11] A method for generating photoacoustic waves using a photoacoustic wave generating structure including: a light-impermeable light reflector that is arranged in contact with the second surface of a sample contact body having a first surface and a second surface opposite the first surface and reflects measurement light; a light absorber that is at least at a position shifted in the planar direction from the light reflector and absorbs excitation pulsed light and converts it into thermal energy; and a light-transmissive photoacoustic wave generator that is arranged in contact with the light absorber and has a thermal expansion coefficient larger than that of the light absorber, the photoacoustic wave generating method including the steps of: irradiating the light absorber with pulsed light from the second surface side to heat the light absorber; and generating photoacoustic waves by thermally expanding the photoacoustic wave generator due to the heat generated by the light absorber.

[12] A method for optically measuring hardness of a sample placed in contact with a first surface by generating photoacoustic waves using a photoacoustic wave generating structure including: a light-impermeable light reflector that is placed in contact with the second surface of a sample contact body having a first surface and a second surface opposite the first surface and reflects measurement light; a light absorber that is at least at a position shifted in a planar direction from the light reflector and absorbs excitation pulsed light and converts it into thermal energy; and a light-transmissive photoacoustic wave generator that is placed in contact with the light absorber and has a thermal expansion coefficient larger than that of the light absorber. the step of placing the photoacoustic wave generating structure in contact with the second surface of the sample contact body; the step of irradiating the light absorber with pulsed light from the second surface side to heat the light absorber; the step of thermally expanding the photoacoustic wave generator by the heat generated by the light absorber to generate a photoacoustic wave; and the step of detecting micro-vibrations from interference between a reflected wave that is reflected at a measurement point on the sample and returns to a specific point on the light reflector when the measurement point is vibrated by the photoacoustic wave and a reference light irradiated to the second surface of the sample contact body. and optically measuring the displacement of the specific point based on the detection result of the micro-vibration, and measuring the hardness of the measurement point of the sample. Effect of the Invention

[0010] As described above in detail, the inventions described in claims 1 to 8 can provide a photoacoustic wave generating sheet capable of efficiently generating photoacoustic waves for sample vibration without using high-intensity pulsed light or a special petri dish.The inventions described in claims 9 to 11 can provide a photoacoustic measurement method and an optical hardness measurement method that can easily perform desired measurements in a non-contact manner at a relatively low cost by using the above-mentioned suitable photoacoustic wave generating sheet. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing the configuration of a measurement system used in a photoacoustic measurement method according to an embodiment. [Diagram 2] FIG. 2 is a plan view of a photoacoustic wave generating seal used in the photoacoustic measurement method according to the embodiment. [Diagram 3] 3 is a cross-sectional view of the photoacoustic wave generating seal of FIG. 2 taken along line AA. [Figure 4] 1 is a schematic diagram for explaining the principle of a photoacoustic measuring method according to an embodiment; [Diagram 5] 1 is a schematic diagram for explaining the principle of a photoacoustic measuring method according to an embodiment; [Figure 6] 1 is a schematic diagram for explaining the principle of a photoacoustic measuring method according to an embodiment; [Figure 7] 1 is a schematic diagram for explaining the principle of a photoacoustic measuring method according to an embodiment; [Figure 8] FIG. 13 is a plan view of a photoacoustic wave generating seal according to another embodiment. [Figure 9] 9 is a cross-sectional view of the photoacoustic wave generating seal of FIG. 8 along line BB. [Figure 10] FIG. 13 is a plan view of a photoacoustic wave generating seal according to another embodiment. [Figure 11] 11 is a cross-sectional view of the photoacoustic wave generating seal of FIG. 10 taken along line CC. [Figure 12]13 is a schematic diagram for explaining the relationship between the thickness t of the bottom portion, the angle θ, and the diameter or diagonal distance D of the light absorber in a photoacoustic wave generating seal according to another embodiment. FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] A measurement system according to this embodiment of the present invention will now be described in detail with reference to Figures 1 to 7. Figure 1 is a schematic diagram showing the configuration of a measurement system used in a photoacoustic measurement method according to this embodiment.

[0013] As shown in FIG. 1, the measurement system of this embodiment is composed of an optical microscope 11 and a photoacoustic measurement unit 31. This measurement system is an apparatus for sealing a plastic petri dish 1 for cell culture to maintain a culture environment and for non-contact measurement of mechanical properties of a cell 2. The photoacoustic measurement unit 31 is a unit for adding a photoacoustic measurement function to an existing optical microscope 11 by retrofitting the optical microscope 11. The optical microscope 11 to which the retrofit photoacoustic measurement unit 31 is attached has basic components such as a microscope body 12, a sample stage 13, an objective lens (light collecting vibration unit) 36, and a reflecting mirror 15. The plastic petri dish 1 containing a cell 2 is placed on the sample stage 13. In this embodiment, the bottom 3 of the plastic petri dish 1 serves as a sample support. The upper surface of the bottom 3 is the support surface 3a side that contacts and supports the cell 2 as a sample. The opposite lower surface is the non-support surface 3b side.

[0014] The objective lens 36 provided on the microscope-side main optical path R1 is configured to focus light on the cell 2 in the plastic petri dish 1 for optical observation. The objective lens 36 as a focusing vibration unit is disposed on the non-support surface 3b side of the bottom 3, which is a sample support, that is, below the bottom 3 in FIG. 1. This objective lens 36 functions as a means for focusing the excitation light L1 on the vibration region P1 on the non-support surface 3b of the bottom 3 and applying vibration to the vibration region P1. The reflecting mirror 15 is provided on the microscope-side main optical path R1. The reflecting mirror 15 reflects the light beam incident from the objective lens 36 side to reach the camera port 14.

[0015] The photoacoustic measurement unit 31 includes a pulsed light source 21, an optical scanner (scanning section) 41, an optical interferometer 51, a control device 61, an input / output interface 63, etc., inside a device body 32. On a main optical path R1 which is a path of a light ray from the pulsed light source 21 to the non-support surface 3b, a lens unit 22 having a pinhole disposed between a pair of lenses, a dichroic mirror 26, and an optical scanner 41 are arranged in this order from the pulsed light source 21 side.

[0016] The pulse light source 21 is a light source for generating excitation light L1 that generates a photoacoustic wave W1. The wavelength of the pulse light generated by this pulse light source 21 is not particularly limited, but may be, for example, about 340 nm to 1200 nm (i.e., a wavelength ranging from the ultraviolet region to the near infrared region), and is set to 532 nm or 1064 nm in this embodiment. As this pulse light source 21, one that generates a very short pulse light is used, for example, one that generates a pulse light with a pulse width of 100 nanoseconds or less is used. Specifically, a pulse laser light source that generates a pulse laser light with a pulse width of about 10 nanoseconds is used. In this embodiment, a pulse laser light source capable of generating a pulse laser light of about 10 nanoseconds is used. Note that a pulse laser light source is preferable as such a pulse light source 21, but instead of the pulse laser light source, for example, a light emitting diode may be used.

[0017] The optical scanner 41 as a scanning unit is a means for moving the vibration region P1 on the non-support surface 3b of the bottom portion 3 at high speed along the surface direction (XY direction) of the non-support surface 3b. In this embodiment, a galvanometer scanner that scans light using a pair of galvanometer mirrors 42 and 43 is used. Note that instead of the galvanometer scanner using the galvanometer mirrors 42 and 43, a MEMS scanner that scans light using, for example, a MEMS mirror may be used.

[0018] The optical interferometer 51 is a device for optically detecting vibrations caused by the photoacoustic wave W1, and is disposed on another optical path (sub-optical path R2) branched off from the main optical path R1. A suitable optical interferometer 51 is, for example, a Michelson interferometer, which is used in this embodiment. The sub-optical path R2 and the main optical path R1 intersect at the dichroic mirror 26. A lens unit 54 having a pinhole disposed between a pair of lenses is disposed between the dichroic mirror 26 and the optical interferometer 51. The optical interferometer 51 has a photoelectric conversion unit 52 and a laser light source 53. The laser light source 53 generates measurement light L2 used for measurement by optical interferometry. Since the measurement light L2 is not for generating the photoacoustic wave W1, light of a wavelength different from the above-mentioned excitation light L1 (excitation light L1 of 532 nm or 1064 nm) is used. In this embodiment, for example, a laser beam in the entire visible light range (a laser beam of about 400 nm to 700 nm (excluding 532 nm)) is used as the measurement beam L2. This measurement beam L2 reaches the non-supporting surface 3b side of the bottom 3 through the objective lens 36, which is a light collecting and exciting unit, and then returns as reflected beam L3. A part of the measurement beam L2 irradiated from the laser beam source 53 is branched on the way and becomes a reference beam (not shown), which is reflected by a reference body (not shown) and then combined with the reflected beam L3. The photoelectric conversion unit 52 receives the combined beam of the reflected beam L3 of the measurement beam L2 reflected and returned by the non-supporting surface 3b side and the reference beam, detects the minute vibration (i.e., minute displacement) of the bottom 3 from the interference between the two, and converts it into an electrical signal. For example, a photodiode or a photomultiplier tube is used as the photoelectric conversion unit 52.

[0019] The control device 61 is a personal computer including, for example, a CPU, a ROM, a RAM, etc., and is electrically connected to the pulse light source 21, the objective lens 36, the scanning unit 41, and the optical interferometer 51. The control device 61 stores a predetermined program, and appropriately controls the operations of the pulse light source 21, the objective lens 36, the scanning unit 41, and the optical interferometer 51 according to the program. The control device 61 serves as a mechanical property value calculation unit. The mechanical property value calculation unit performs a calculation to calculate a mechanical property value at the measurement point P2 based on data from the optical interferometer 51. In the control device 61 of this embodiment, for example, hardness, elasticity, and viscosity are calculated based on a program for measuring hardness, elasticity, and viscosity. The input / output interface 63 is electrically connected to the control device 61 and is also electrically connected to a display device, an input device, etc., not shown, outside the device.

[0020] Next, a photoacoustic wave generating sheet (photoacoustic wave generating tape) 71 used in the photoacoustic measurement method will be described. Fig. 2 is a plan view of the photoacoustic wave generating tape 71, and Fig. 3 is a cross-sectional view taken along line AA thereof. This photoacoustic wave generating tape 71 is a member used when performing photoacoustic measurement on a cell 2 arranged in contact with the supporting surface 3a (i.e., the inner surface) of the bottom 3, and is arranged in contact with the non-supporting surface 3b (i.e., the outer surface) of the bottom 3. Note that this photoacoustic wave generating tape 71 is a sheet-like laminate having flexibility overall, and can be attached to and peeled off from the non-supporting surface 3b of the bottom 3.

[0021] The photoacoustic wave generating tape 71 of this embodiment includes a light reflector 73, a light absorber 74, a photoacoustic wave generator 72, an adhesive layer 76, and a protective layer 77. The shape of the photoacoustic wave generating tape 71 in plan view is not particularly limited and may be any shape, but in this embodiment, for example, the shape is rectangular. The photoacoustic wave generating tape 71 includes a tape body including the light reflector 73, the light absorber 74, the photoacoustic wave generator 72, and the adhesive layer 76, and a protective layer 77 that covers and protects one side of the tape body. The protective layer 77 is also a flexible base material that supports the tape body, and is formed of, for example, a paper or resin sheet material. The protective layer 77 can be peeled off from the adhesive layer 76, and is removed by peeling it off from the tape body when the photoacoustic wave generating tape 71 is used. The adhesive layer 76 is provided on the entire area of ​​one side of the protective layer 77. The adhesive layer 76 is, for example, a layer formed from a transparent adhesive having optical transparency, and specifically, a layer formed from, for example, a rubber-based adhesive, an acrylic-based adhesive, a silicone-based adhesive, or the like.

[0022] The light reflector 73 in the photoacoustic wave generating tape 71 is a portion for reflecting measurement light (i.e., light emitted by an optical interferometer, continuous wave or pulse wave light), and is formed on the adhesive layer 76 using a material capable of reflecting light. In this embodiment, the light reflector 73 is a plurality of metal layers formed in a rectangular band shape in a plan view, and is arranged so as to be parallel to each other and at equal intervals. The area ratio of the light reflector 73 in the photoacoustic wave generating tape 71 is not particularly limited and is set arbitrarily. For example, the light reflector 73 is formed so as to occupy an area of ​​less than 50% of the area of ​​the photoacoustic wave generating tape 71, and is preferably formed so as to occupy an area of ​​less than 40%. When the area ratio of the light reflector 73 is set in this manner, the sample 2 can be easily observed with an optical microscope using transmitted light, and as a result, for example, the position where the sample 2 is present can be easily grasped before photoacoustic measurement. Note that if the area ratio of the light reflector 73 is too high, the light is easily blocked by the light reflector 73, and the amount of transmitted light may be reduced.

[0023] The metal layer forming the light reflector 73 is not particularly limited as long as it can reflect at least a part of the incident light, and any metal layer can be used. Examples of such metal layers include a copper layer, an aluminum layer, a nickel layer, a tin layer, and an iron layer. The light reflector 73 made of a metal layer may be formed by any thin film formation method, and examples of such methods include an etching method, a plating method, and a printing method. Here, for example, a procedure of attaching an aluminum foil and then etching is adopted. Alternatively, a procedure of attaching an aluminum foil cut to a predetermined size and shape in advance to a predetermined position may be adopted.

[0024] The light absorber 74 is provided as a portion that absorbs the pulsed light for excitation and converts it into thermal energy. The shape and size of the light absorber 74 are not particularly limited, but the light absorber 74 in this embodiment is rectangular and is formed at equal intervals at a plurality of locations on the light reflector 73 that is rectangular strip-shaped. That is, the light absorber 74 is laminated on the light reflector 73 (in other words, it is present at a position that is not shifted from the light reflector 73 in the sheet surface direction), but it can be understood that it is also present at a position shifted from the light reflector 73 in the sheet surface direction in a plan view. In addition, the light absorber 74 in this embodiment is a fine particle dispersion layer in which inorganic fine particles that absorb light are dispersed, and specifically, carbon fine particles, gold nanoparticles, etc. are dispersed. Here, the fine particle dispersion layer that is the light absorber 74 is formed by using a method of printing black ink in which carbon black is dispersed.

[0025] The acoustic wave generator 72 constituting the photoacoustic wave generating tape 71 is provided as a portion that generates a photoacoustic wave based on the irradiation of pulsed light to the light absorber 74. The acoustic wave generator 72 is arranged in contact with the light reflector 73 and the light absorber 74, and is a layer made of a light-transmitting material having a thermal expansion coefficient larger than that of the light absorber 74. The photoacoustic wave generator 72 is provided over the entire area of ​​the sheet to cover the light reflector 73 and the light absorber 74. Specifically, in this embodiment, the photoacoustic wave generator 72 is a transparent resin layer formed with a larger area and a uniform thickness than the light reflector 73 and the light absorber 74. Such a transparent resin layer can be formed using, for example, a cured product of a two-component curable resin.

[0026] When pulsed light is irradiated onto the light absorber 74 covered with the photoacoustic wave generator 72, the light absorber 74 absorbs light energy and converts it into thermal energy, thereby increasing its own temperature. Then, the adjacent region P4 (see Figs. 5 and 6) in the photoacoustic wave generator 72, which is immediately adjacent to the light absorber 74, is instantly heated, and its volume instantly increases due to thermal expansion. Due to this photoacoustic effect, a longitudinal or transverse photoacoustic wave is generated with the adjacent region P4 as an oscillation source. Incidentally, when the photoacoustic wave generator 72 is formed, it is possible to increase the efficiency of generating photoacoustic waves by 10 times or more compared to when the photoacoustic wave generator 72 is not formed.

[0027] As described above, the light reflectors 73 and the light absorbers 74 in this embodiment are each formed in a plurality of numbers. The gaps C1 between the plurality of light reflectors 73 and the gaps C2 between the plurality of light absorbers 74 are preferably set to be smaller than the size of the cells 2 as the sample. In this embodiment, for example, when a human cell 2 having a diameter of about 10 μm to 30 μm is used as the sample, it is preferable that both the gaps C1 and C2 are set to a value smaller than that, specifically, about 1 μm to 10 μm. The size of the light absorber 74 in a plan view is preferably larger than the wavelength of the photoacoustic wave generated. In addition, the thicknesses of the light absorber 74 and the photoacoustic wave generator 72 are preferably set to be equal to or larger than the wavelength of the photoacoustic wave generated. For example, when a pulsed laser beam having a pulse width of about 10 nanoseconds is used, it is known that the center frequency of the generated photoacoustic wave is about 20 MHz. In this case, the wavelength of the photoacoustic wave is about 100 μm. Therefore, the size of the light absorber 74 in plan view is preferably set to be greater than 100 μm, and the thickness of the light absorber 74 and the photoacoustic wave generator 72 is preferably set to be 100 μm or greater.

[0028] Next, the procedure of photoacoustic measurement using this measurement system will be described. Figs. 4 to 7 are schematic diagrams for explaining the principle of the photoacoustic measurement method. First, a plastic petri dish 1 containing cells 2 as a sample is prepared, and a photoacoustic wave generating tape 71 is attached to the non-supporting surface 3b of the bottom 3 (see Fig. 4). Specifically, the protective layer 77 is removed from the photoacoustic wave generating tape 71 to leave only the tape body, and then the tape body is attached to the bottom 3 so that the exposed adhesive layer 76 side is brought into surface contact with the non-supporting surface 3b. At this time, the photoacoustic wave generating tape 71 does not necessarily have to be disposed over the entire area of ​​the bottom 3, and may be disposed, for example, only in the center portion.

[0029] Next, the user places the plastic petri dish 1 with the photoacoustic wave generating tape 71 attached on the sample stage 13. At this time, the objective lens 36 is driven and adjusted so that the focal point is located near the non-support surface 3b of the bottom part 3. When performing photoacoustic measurement, the cells 2 to be measured are usually identified in advance by optical microscope observation using a conventionally known method.

[0030] Next, the user inputs an instruction to start photoacoustic measurement using an input device (not shown). Then, the control device 61 performs control to switch from the optical microscope mode to the photoacoustic measurement mode. Furthermore, when information instructing the start of measurement is input from the input device, the control device 61 controls the pulse light source 21 to repeatedly emit a pulse laser light (excitation light L1) (see FIG. 5). In addition, while the pulse light source 21 repeatedly emits the excitation light L1, the control device 61 drives the laser light source 53 of the optical interferometer 51 to continuously emit a laser light that is the measurement light L2. In this embodiment, the irradiation area of ​​the measurement light L2 is set to be smaller than the irradiation area of ​​the excitation light L1.

[0031] The excitation light L1 generated by the pulse light source 21 passes through the lens unit 22, the dichroic mirror 26, the optical scanner 41, and the objective lens 36, and is collected in the vibration region P1 near the non-support surface 3b of the bottom 3. At this time, the control device 61 drives and controls the optical scanner 41 so that the excitation light L1 is collected at a predetermined scanning position. Then, the excitation light L1 collected in the vibration region P1 excites vibration in the vibration region P1 by photoacoustic action (see FIG. 6). Specifically, in this embodiment, the spot size of the excitation light L1 is adjusted so that multiple (two in FIG. 5) light absorbers 74 are included in the vibration region P1. When the excitation light L1 is irradiated, each light absorber 74 converts the light energy of the excitation light L1 into thermal energy and increases in temperature. Then, the adjacent region P4 in the photoacoustic wave generator 72 is instantly heated, and the volume is instantly enlarged by thermal expansion, and as a result, photoacoustic waves W1 are generated with the two adjacent regions P4 as oscillation sources, respectively.

[0032] The photoacoustic waves W1 thus generated at two locations gradually spread radially from the adjacent region P4 as the center, and reach the support surface 3a of the bottom 3. The photoacoustic waves W1 then converge at the midpoint between the two adjacent regions P4 in a plan view (i.e., the measurement point P2 of the sample 2), and are thus in a constructive state. As a result, the photoacoustic waves W1 are reflected at the measurement point P2 of the sample 2 and return to a specific point P5 on the light reflector 73 (see FIG. 7). As a result, the bottom 3 vibrates slightly, and the surface of the light reflector 73 is slightly displaced.

[0033] The measurement light L2 is irradiated to a specific point P5 on the light reflector 73 and is reflected at that position. The reflected measurement light L2 (i.e., reflected light L3) travels back along the main optical path R11 to the dichroic mirror 26, where it is redirected to the sub-optical path R12 and combined with the reference light. This combined light is received by the photoelectric conversion unit 52 of the optical interferometer 51 and converted into an electrical signal. That is, the micro-vibration of the bottom 3 is detected from the interference between the reflected light L3 and the reference light, and converted into an electrical signal. At this time, the control device 61 stores the electrical signal from the photoelectric conversion unit 52 in accordance with the emission timing of the excitation light L1. The control device 61 then performs a calculation to calculate the mechanical property value (hardness, elasticity, viscosity) at the measurement point P2 based on the detection result of the micro-vibration obtained from the previously stored electrical signal. The mechanical property value calculated in this manner is output to the display device via the input / output interface 63 and displayed in a predetermined manner.

[0034] Therefore, according to this embodiment, the following effects can be obtained.

[0035] (1) The photoacoustic wave generating tape 71 of this embodiment is arranged in contact with the non-support surface 3b of the bottom 3 of the plastic petri dish 1, and includes a light reflector 73, a light absorber 74, and a photoacoustic wave generator 72. The light absorber 74 is located at a position shifted from the light reflector 73 in the sheet surface direction, and absorbs pulsed light (excitation light L1) and converts it into thermal energy. The light-transmitting photoacoustic wave generator 72 is arranged in contact with the light absorber 74 and has a larger thermal expansion coefficient than the light absorber 74. Therefore, even when a relatively weak pulsed light L1 is irradiated onto a general plastic petri dish 1, the photoacoustic effect can be reliably obtained. Therefore, the photoacoustic wave W1 for sample vibration can be efficiently generated without using a high-intensity pulsed light L1 or a special petri dish. In particular, according to the embodiment, a large pulsed light generating unit is not required, so not only can the entire device be formed compactly, but also the cost of the device can be easily reduced.

[0036] (2) The photoacoustic wave generating tape 71 of this embodiment is flexible as a whole and can be attached to and peeled off from the non-supporting surface 3b of the bottom 3. The photoacoustic wave generating tape 71 configured in this manner can be easily installed on an existing plastic petri dish 1 when necessary, and can be easily removed when no longer necessary. Therefore, it is not necessary to use a special petri dish for photoacoustic measurement. In addition, since it is possible to use a general plastic petri dish 1, the cost of performing photoacoustic measurement can be reduced. Furthermore, this photoacoustic wave generating tape 71 includes a light-transmitting adhesive layer 76 formed on one side of the sheet, and a protective layer 77 formed so as to be peelable so as to cover the adhesive layer 76. Therefore, the adhesive layer 76 is protected by the protective layer 77 until use, and as a result, the adhesive force is prevented from decreasing. In addition, the risk of dust adhering to the surface of the adhesive layer 76 is reduced, and the deterioration of the photoacoustic measurement accuracy caused by the adhesion of dust to the non-supporting surface 3b of the bottom 3 is also prevented.

[0037] (3) In the photoacoustic wave generating tape 71 of this embodiment, the light reflector 73 is formed so as to occupy an area less than 50% of the area of ​​the sheet. In addition, the photoacoustic wave generator 72 and the adhesive layer 76, which are formed in a relatively large area, are optically transparent. This allows light to pass through the cells 2 to a certain extent, making it possible to simultaneously observe the cells 2 using transmitted light with an optical microscope and measure the hardness, etc., of the cells 2 using photoacoustic measurement.

[0038] (4) In the photoacoustic wave generating tape 71 of this embodiment, not only are the light reflectors 73 and the light absorbers 74 formed in multiple numbers, but they are also regularly arranged along the planar direction of the sheet. Also, as shown in Figs. 3 to 7, the gaps C1 between the multiple light reflectors 73 and the gaps C2 between the multiple light absorbers 74 are both set to be smaller than the size of the cell 2. With this configuration, photoacoustic waves W1 are generated with the adjacent multiple adjacent regions P4 as oscillation sources, and these photoacoustic waves W1 are focused at the measurement point P2 of the cell 2. Therefore, a photoacoustic wave W1 that is reinforced can be obtained, and as a result, the measurement sensitivity can be improved. Also, since the light reflectors 73 and the light absorbers 74 are smaller than the cell 2, it is possible to measure the hardness at multiple measurement points in the cell 2 by scanning the laser light L1 in the planar direction.

[0039] (5) In this embodiment, a method for optically measuring the stiffness of the cell 2 by generating a photoacoustic wave W1 using the photoacoustic wave generating tape 71 having the above configuration is disclosed. In the first step, the photoacoustic wave generating tape 71 (photoacoustic wave generating structure) is placed in contact with the non-supporting surface 3b of the bottom 3. In the next step, pulsed light L1 is irradiated onto the light absorber 74 from the non-supporting surface 3b side to heat the light absorber 74. In the next step, the photoacoustic wave generator 72 is thermally expanded by the heat generated by the light absorber 74 to generate a photoacoustic wave W1. In the next step, micro-vibrations are detected from the interference between the reflected wave L3 returning to the specific point P5 on the light reflector 73 and the reference light irradiated onto the non-supporting surface 3b of the bottom 3. In the next step, the displacement of the specific point P5 is optically measured based on the detection result of the micro-vibrations, and the stiffness of the measurement point P2 of the cell 2 is measured. According to this optical hardness measurement method, the displacement can be measured by indirectly applying vibration to the cell 2 using the photoacoustic effect, rather than directly applying an external force to the cell 2 to measure the displacement. In other words, the plastic petri dish 1 for cell culture is sealed to maintain the culture environment, and vibration and measurement are performed in this state in a non-contact manner, thereby measuring the hardness of the cell 2. Therefore, there is no need to worry about contamination or damage of the cell 2 during culture. As described above, according to the optical hardness measurement method of this embodiment, the hardness of the desired cell 2 can be measured in a non-contact manner at a relatively low cost and easily.

[0040] (6) Furthermore, in the optical stiffness measurement method of this embodiment, the excitation light L1 is not focused on the cell 2 (measurement point P2) and vibrated, but is focused on a vibration region P1 located in front of the measurement point P2 and vibrated. As a result, it is no longer necessary to irradiate the cell 2 itself with the pulsed laser light L1, which is the excitation light L1, and therefore a rise in the temperature of the cell 2 can be avoided and the activity of the cell 2 can be maintained.

[0041] The embodiment of the present invention is merely an example, and may be modified as appropriate without departing from the spirit of the invention.

[0042] In the photoacoustic wave generating tape 71 of the above embodiment, a plurality of rectangular strip-shaped reflectors 73 are arranged in parallel and at equal intervals, and rectangular light absorbers 74 are arranged at equal intervals at a plurality of locations on the reflectors 73, but the present invention is not limited to this. For example, the photoacoustic wave generating tape 71A of another embodiment shown in Figs. 8 and 9 may be configured. In this photoacoustic wave generating tape 71A, a plurality of light reflectors 73 each having a circular shape in a plan view are formed at equal intervals. In addition, annular light absorbers 74 are formed so as to surround each light reflector 73. Note that adjacent annular light absorbers 74 partially overlap each other. The light absorbers 74 are located at positions shifted from the light reflectors 73 in the sheet surface direction, and are not formed to overlap the light reflectors 73. Alternatively, the photoacoustic wave generating tape 71B of another embodiment shown in Figs. 10 and 11 may be configured. In this photoacoustic wave generating tape 71B, a plurality of light reflectors 73 each having a circular shape in a plan view are formed at equal intervals. Moreover, a hexagonal light absorber 74 is formed so as to surround each light reflector 73. Adjacent hexagonal light absorbers 74 partially overlap each other. In these other embodiments, the diameter of the light reflector 73 is larger than the width of the light absorber 74, and the diameter or diagonal distance (D) of the light absorber 74 is larger than the diameter of the light reflector 73. The diameter of the light reflector 73 is set to be larger than the spot size of the measurement light L2. Here, if the thickness of the bottom part 3, which is the sample contact body, is t and the angle of the part shown in FIG. 12 is θ, the relationship "D=2×t×tan θ" is established. From the viewpoint of the generation efficiency of the photoacoustic wave W1, etc., it is preferable to set θ within the range of 30° to 60°. Therefore, it is preferable to set D to be within the range of "2×t×tan 30° to 2×t×tan 60°".

[0043] In the above embodiment, the photoacoustic wave generating tape 71 is flexible as a whole, but is not limited to this and may of course be made of a rigid body that does not have flexibility.

[0044] In the above embodiment, the photoacoustic wave generating tape 71 formed separately from the plastic petri dish 1 is attached to the plastic petri dish 1, but the present invention is not limited to this. For example, the photoacoustic wave generating structure may be formed by directly printing or applying various materials to the plastic petri dish 1. In other words, the photoacoustic wave generating structure does not necessarily have to be in a sheet shape.

[0045] In the above embodiment, the sample is the cell 2, and the sample support is the bottom 3 of the plastic petri dish 1 for cell culture, but this is not limiting. For example, the sample may be an in vivo tissue, and the sample contact body may be a cap or cover (tip placement member) provided at the tip of an endoscope. In other words, the photoacoustic wave generating tape 71 of the present invention may be used for medical purposes such as checking the hardness of an organ during surgery. Of course, measurement may be performed using a non-living object as a sample instead of tissue in a living body. Of course, something other than the plastic petri dish 1 or the tip placement member may be used as the sample support.

[0046] In the above embodiment, the optical scanner 41 is used as the scanning unit. However, instead of an optical scanning means such as the optical scanner 41, a mechanical scanning means such as an XY table may be used.

[0047] The light absorber 74 does not necessarily have to be formed on and in contact with the light reflector 73. In other words, the light absorber 74 may be spaced apart from the light reflector 73 in the thickness direction of the sheet and disposed so as to sandwich the photoacoustic wave generator 72 therebetween. [Explanation of symbols]

[0048] 2: Cells as samples 3: Bottom of a plastic petri dish as a sample contact body 3a: Support surface as the first surface 3b: Non-supporting surface as the second surface 71, 71A, 71B: Photoacoustic wave generating tape as a photoacoustic wave generating structure (photoacoustic wave generating sheet) 72: Transparent resin layer as a photoacoustic wave generator 73: Metal layer as a light reflector 74: Fine particle dispersion layer as a light absorber 76: Adhesive layer 77: Protective layer C1: Gap (between multiple light reflectors) C2: Gaps (between multiple light absorbers) L1: Pulsed light L2: Reference beam L3: Reflected wave P2: Measurement point (of the sample) P5: Specific point (on the light reflector)

Claims

1. A photoacoustic wave generating sheet is arranged in contact with a second surface on an opposite side to a first surface of a sample contact body, in order to perform photoacoustic measurement on a sample arranged in contact with the first surface, a light reflector that reflects the measurement light; a light absorber that is at least located at a position shifted from the light reflector in a surface direction of the sheet and absorbs the pulsed light for excitation and converts it into thermal energy; a light-transmitting photoacoustic wave generator that is arranged in contact with the light absorber and has a thermal expansion coefficient larger than that of the light absorber; A photoacoustic wave generating sheet comprising:

2. The photoacoustic wave generating sheet according to claim 1, characterized in that the photoacoustic wave generator is a transparent resin layer that covers the light reflector and the light absorber, and is formed with a larger area and uniform thickness than them.

3. The light reflector is a metal layer, The light absorber is a fine particle dispersion layer in which inorganic fine particles that absorb light are dispersed. The photoacoustic wave generating sheet according to claim 2 .

4. The photoacoustic wave generating sheet according to claim 3 , wherein the photoacoustic wave generating sheet is a photoacoustic wave generating tape that is flexible as a whole and can be attached to and peeled off from the first surface of the sample contact body.

5. A light-transmitting adhesive layer formed on one side of the sheet; a protective layer formed in a peelable manner so as to cover the adhesive layer; The photoacoustic wave generating sheet according to claim 1 , further comprising:

6. 5. The photoacoustic wave generating sheet according to claim 1, wherein the light reflector is formed so as to occupy an area less than 50% of the area of ​​the photoacoustic wave generating sheet.

7. A plurality of the light reflectors and a plurality of the light absorbers are formed, The gaps between the plurality of light reflectors and the gaps between the plurality of light absorbers are both smaller than the size of the sample.

5. The photoacoustic wave generating sheet according to claim 1, wherein the first and second electrodes are arranged in a first direction.

8. The size of the optical absorber is larger than the wavelength of the generated photoacoustic wave, The thickness of each of the light absorber and the photoacoustic wave generator is equal to or greater than the wavelength of the photoacoustic wave to be generated.

5. The photoacoustic wave generating sheet according to claim 1, wherein the first and second electrodes are arranged in a first direction.

9. 5. The photoacoustic wave generating sheet according to claim 1, wherein the sample contact member is a bottom of a plastic petri dish, and the sample is a cell.

10. 5. The photoacoustic wave generating sheet according to claim 1, wherein the sample contact body is a tip arrangement member provided at the tip of an endoscope, and the sample is a living tissue.

11. a sample contact body having a first surface and a second surface opposite the first surface, the sample contact body being disposed in contact with the second surface; a light reflector that reflects the measurement light; a light absorber that is at least located at a position shifted from the light reflector in a planar direction and absorbs the pulsed light for excitation and converts it into thermal energy; a light-transmitting photoacoustic wave generator that is arranged in contact with the light absorber and has a thermal expansion coefficient larger than that of the light absorber; A method for generating photoacoustic waves using a photoacoustic wave generating structure comprising: irradiating the light absorber with pulsed light from the second surface side to heat the light absorber; generating a photoacoustic wave by thermally expanding the photoacoustic wave generator due to heat generated by the light absorber; A photoacoustic wave generating method comprising:

12. a sample contact body having a first surface and a second surface opposite the first surface, the sample contact body being disposed in contact with the second surface; a light reflector that reflects the measurement light; a light absorber that is at least located at a position shifted from the light reflector in a planar direction and absorbs the pulsed light for excitation and converts it into thermal energy; a light-transmitting photoacoustic wave generator that is arranged in contact with the light absorber and has a thermal expansion coefficient larger than that of the light absorber; A method for optically measuring the hardness of a sample placed in contact with the first surface by generating a photoacoustic wave using a photoacoustic wave generating structure comprising: placing the photoacoustic wave generating structure in contact with the second surface of the sample contact body; irradiating the light absorber with pulsed light from the second surface side to heat the light absorber; generating a photoacoustic wave by thermally expanding the photoacoustic wave generator due to heat generated by the light absorber; a step of detecting micro-vibrations from interference between a reflected wave that is reflected at a measurement point on the sample and returns to a specific point on the optical reflector when the measurement point on the sample is vibrated by the photoacoustic wave and a reference light irradiated on the second surface of the sample contact body; a step of optically measuring the displacement of the specific point based on a detection result of the micro-vibration and measuring the hardness of the measurement point of the sample; An optical hardness measurement method comprising:

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