JP2020134655A - Observation device, method for observation, microscope device, and endoscope device - Google Patents

Abstract

To provide an observation device which can detect a photoacoustic signal based on a two-photon excitation rapidly and with a simple structure.SOLUTION: An observation device 50 includes: a pulse laser 10 for generating light L; an irradiation optical system 20 for irradiating a sample T with a light L; and a detector 40 for detecting a signal in association with a reaction of the sample T caused by irradiation with the light L. The irradiation unit 20 includes a switch unit 51 for switching a first mode for generating one-photon excitation and two-photon excitation in the sample T by irradiating a focus surface with light LA with a first intensity distribution and a second mode for generating at least one one-photon excitation in the sample T by irradiating the focus surface with light LB with a second intensity distribution.SELECTED DRAWING: Figure 1

Description

The present invention relates to an observation device, an observation method, a microscope device, and an endoscopic device.

Photoacoustic microscopy (PAM, photoacoustic microscopy), which can image the absorption distribution of a sample without using reagents by detecting photoacoustic (PA, photoacoustic) waves generated by light absorption, is widely used in the medical field, for example. Has been done.

As an example of such a photoacoustic microscope, a two-photon excitation photoacoustic microscope has been proposed (see, for example, Non-Patent Document 1). The photoacoustic microscope proposed in Non-Patent Document 1 uses a laser having a variable pulse width. In this photoacoustic microscope, a photoacoustic signal is acquired by light having a pulse width of about femtoseconds, and a photoacoustic signal is acquired by light having a pulse width of about picoseconds. Photoacoustic signals due to light with a pulse width of about picoseconds are dominated by photoacoustic signals due to one-photon excitation, and photoacoustic signals for light with a pulse width of about femtoseconds are light due to one-photon excitation and two-photon excitation. The acoustic signal is dominant. Therefore, by subtracting the photoacoustic signal of light having a pulse width of about picoseconds from the photoacoustic signal of light having a pulse width of about femtoseconds, only the photoacoustic signal due to diphoton excitation can be extracted.

However, the photoacoustic microscope disclosed in Non-Patent Document 1 described above uses a special laser having a variable pulse width, and it takes time to change the pulse width.

Yoshihisa Yamaoka et. Al., “Photoacoustic microscopy using ultrashort pulses with two different pulse durations”, OPTICS EXPRESS (US), 2014, Vol.22, No.14, p.17063-17072

The observation device of the present invention has a light source that generates light, an irradiation optical system that irradiates the sample with light, and a detection unit that detects a signal associated with the reaction of the sample due to the irradiation of light. The system irradiates the sample with light having a first intensity distribution on the focal plane in a first mode of generating one-photon excitation and two-photon excitation, and light having a second intensity distribution on the focal plane. This has a switching unit for switching between the second mode in which at least one photon excitation is generated in the sample.

The observation method of the present invention has a first mode in which one photon excitation and two photon excitation are generated in a sample by irradiating light having a first intensity distribution on the focal plane, and a second intensity distribution on the focal plane. It includes a step of switching between a second mode in which at least one photon excitation is generated in the sample by irradiating the sample with light, and a step of detecting a signal accompanying the reaction of the sample due to the irradiation of light.

It is a block diagram which shows the schematic structure of the photoacoustic microscope to which the observation apparatus which is 1st Embodiment is applied. It is the schematic which showed the vicinity of the sample enlarged in the photoacoustic microscope which concerns on 1st Embodiment. It is a waveform diagram for demonstrating an example of the operation of the photoacoustic microscope which concerns on 1st Embodiment. It is a block diagram which shows the schematic structure of the photoacoustic microscope to which the observation apparatus which is 2nd Embodiment is applied. It is a figure for demonstrating the principle used for the observation apparatus which is a 2nd Embodiment. It is a figure which shows an example of the profile of the light which irradiates a sample by the observation apparatus which is 2nd Embodiment. It is a figure which shows an example of the phase of the pupil function of the objective lens in the photoacoustic microscope which concerns on 2nd Embodiment. It is a figure which shows an example of the profile of the light in the focal plane of the light which irradiates a sample by the observation apparatus which is 2nd Embodiment.

Hereinafter, embodiments will be described with reference to the drawings.

(First Embodiment)
A photoacoustic microscope to which the observation device according to the first embodiment is applied will be described with reference to FIGS. 1 and 2.

FIG. 1 is a block diagram showing a schematic configuration of a photoacoustic microscope to which the observation device according to the first embodiment is applied, and FIG. 2 is an enlarged view of the vicinity of a sample of the photoacoustic microscope according to the first embodiment. It is a schematic diagram shown.

In these figures, the photoacoustic microscope 1 according to the present embodiment has a pulse laser 10 as a light source, an irradiation optical system 20, and a detection unit 40.

The irradiation optical system 20 includes a half mirror 21, a first acousto-optic modulator (hereinafter referred to as a first AOM) 22, a mirror 23, an optical fiber 24, and a second acousto-optic element (hereinafter referred to as a first AOM). It has 25 (referred to as AOM of 2), a mirror 26, a half mirror 27, a dichroic mirror 28, a galvano scanner (scanning unit) 29, an objective lens 30, and a function generator 31.

The half mirrors 21 and 27 may be replaced with a polarizing beam splitter. In this case, in order to equalize the amount of light of the pulsed light LA and the pulsed light LB, which will be described later, it is desirable to install a half-wave plate after the emission of the pulsed laser 10 so that the polarized light is linearly polarized at 45 °. Further, in order to equalize the polarizations of the pulsed light LA and the pulsed light LB, it is desirable to install a polarizer in the optical path after combining the two lights and set the polarization axis to 45 °.

The pulse laser 10 emits a laser beam L having a predetermined pulse width. The pulse width of the light L generated by the pulse laser 10 is determined from the viewpoint of whether or not the pulsed light L can generate both one-photon excitation and two-photon excitation in the sample T. In the photoacoustic microscope 1 according to the present embodiment, the pulse width of the light L emitted from the pulse laser 10 is set to 200 fs as an example.

The optical path of the pulsed light L emitted from the pulsed laser 10 is branched in two directions by the half mirror 21. One LA of the pulsed light branched by the half mirror 21 is incident on the first AOM 22.

The first AOM 22 has an ultrasonic element bonded to the side surface of an acoustic optical medium. When an electric signal of a predetermined frequency f output from the function generator 31 is input to an AOM driver (not shown) and the ultrasonic element is driven by a drive signal from the AOM driver, the ultrasonic sound output from the ultrasonic element is acoustic. Propagates in the optical medium. This ultrasonic wave causes a change in the refractive index in the acoustic optical medium, and the acoustic optical medium acts as a so-called diffraction grating. Therefore, the pulsed light LA incident on the first AOM 22 is diffracted by the first AOM 22. In the irradiation optical system 20 of the present embodiment, the primary diffracted light from the first AOM 22 is used as an output.

The primary diffracted light LA emitted from the first AOM 22 passes through the half mirror 27 and the dichroic mirror 28 and is incident on the galvano scanner 29.

The dichroic mirror 28 passes the pulsed light LA. Further, as will be described later, the dichroic mirror 28 reflects light having a fluorescence wavelength emitted by the sample T when the sample T is labeled with a fluorescent reagent. One surface of the dichroic mirror 28 is coated with a dielectric coating having such a wavelength dependence.

The fluorescence reflected by the dichroic mirror 28 is input to the photomultiplier tube 33 through the optical filter 32. The output of the photomultiplier tube 33 is input to the information processing device 41 of the detection unit 40. The details of the operation of the information processing device 41 will be described later.

The galvano scanner 29 has two reflectors (galvano mirrors) 29a and 29b and a drive unit (not shown) for independently driving the reflectors 29a and 29b. By driving the reflectors 29a and 29b by the driving unit, the pulsed light LA can be scanned at an arbitrary position on a plane orthogonal to the optical axis.

The light LA scanned at a predetermined position is focused and incident on the predetermined position of the sample T (for example, the spot SP1 shown in FIG. 1) by the objective lens 30.

On the other hand, the other LB of the pulsed light branched by the half mirror 21 is guided in a predetermined direction by the mirror 23 and is incident on the optical fiber 24 which is the pulse width extension portion. The light propagating in the optical fiber 24 is incident on the second AOM 25. When the pulsed light LB propagates in the optical fiber 24, its pulse width is extended under the influence of wavelength dispersion. A glass rod may be installed before the light is incident on the optical fiber 24 to extend the pulse width to some extent.

The pulse width of the optical LB extended by the optical fiber 24 is determined from the viewpoint of whether or not at least one photon excitation can be generated in the sample T by the pulsed light LB having the extended pulse width. In the photoacoustic microscope 1 according to the present embodiment, as an example, the pulse width of the optical LB emitted from the optical fiber 24 is 200 ps. Therefore, the structure, material, and total length of the optical fiber 24 are determined so that light having a pulse width of 200 fs is incident and light LB having a pulse width of 200 ps is emitted. The pulse width of the extended optical LB may be made variable by making the total length of the optical fiber 24 variable.

The pulsed light LB emitted from the optical fiber 24 is incident on the second AOM 25. When an electric signal having a predetermined frequency f is input from the function generator 31 to an AOM driver (not shown) and a drive signal from the AOM driver is applied to the second AOM 25, the second AOM 25 acts as a diffraction grating. Also in the irradiation optical system 20 of the present embodiment, the primary diffracted light from the second AOM 25 is used as an output.

The optical path of the primary diffracted light LB emitted from the second AOM 25 is guided in a predetermined direction by the mirror 26, and is incident on the half mirror 27. When the light LB is reflected by the half mirror 27 and emitted, the light LB is incident at a position that shares the optical axis with the light LA emitted from the first AOM 22.

The light LB reflected and emitted by the half mirror 27 is incident on the dichroic mirror 28 in the same manner as the light LA emitted through the half mirror 27.

The light LB that has passed through the dichroic mirror 28 is incident on the galvano scanner 29. The light LB scanned at a predetermined position by the galvano scanner 29 is focused by the objective lens 30 at a predetermined position of the sample T, which is substantially the same as the light LA, and is incident.

The detection unit 40 includes a transducer 42 and a photoacoustic detection unit 43 of the information processing device 41.

As shown in FIG. 2, in the photoacoustic microscope 1 according to the present embodiment, the sample T is immersed in the medium M through which light and photoacoustic waves can propagate. The medium M is not particularly limited as long as it can propagate light and photoacoustic waves, but in the photoacoustic microscope 1 according to the present embodiment, water is used as the medium M in consideration of ease of handling. There is.

The light LA and LB focused by the objective lens 30 and incident on the predetermined position of the sample T (for example, the spot SP1 shown in FIG. 1) generate a photoacoustic wave W at this predetermined position due to the photoacoustic effect. The transducer 42 receives the photoacoustic wave W generated from the sample T and propagates through the medium M, and outputs an electric signal corresponding to the intensity of the received photoacoustic wave W. Although it depends on the physical properties of the sample T, the photoacoustic wave W is usually a sound wave in the ultrasonic band. Therefore, it is desirable that the transducer 42 is an ultrasonic transducer. Since the materials and the like constituting the ultrasonic transducer are well known, the description thereof is omitted here.

Here, as described above, the light LA emitted from the first AOM 22 has the same pulse width as the light L emitted from the pulse laser 10. That is, the optical LA has a pulse width of about femtoseconds. Since the peak intensity of the light LA having such an extremely short pulse width is sufficiently large, a photoacoustic wave W associated with the two-photon excitation is generated in the sample T irradiated with the light LA in addition to the one-photon excitation. This is the first mode. Further, the light LA having an ultrashort pulse width is light having a first intensity distribution on the focal plane.

On the other hand, the pulse width of the optical LB emitted from the second AOM 25 is extended by the optical fiber 24. As the pulse width is extended by the optical fiber 24, the peak intensity of the optical LB becomes lower than that of the optical LA. Therefore, the photoacoustic wave W associated with the one-photon excitation is generated in the sample T irradiated with the light LB. This is the second mode. Further, the light LB is light having a second intensity distribution on the focal plane.

The output signal of the transducer 42 is input to the information processing device 41. The information processing device 41 has computing power, for example, a personal computer or the like. The information processing device 41 includes a photoacoustic detection unit 43, an image processing unit (image generation unit) 44, and a control unit 45.

The photoacoustic detection unit 43, which forms a part of the detection unit 40, detects the photoacoustic signal of the sample T based on the output signal from the transducer 42. Further, the photoacoustic detection unit 43 extracts only the photoacoustic signal generated by two-photon excitation from the photoacoustic signal based on the optical LA and the photoacoustic signal based on the optical LB. A method for extracting only the photoacoustic signal generated by two-photon excitation will be described later.

The image processing unit 44 receives the photoacoustic signal obtained from the predetermined position of the sample T from the photoacoustic detection unit 43. Then, the image processing unit 44 generates an image signal that is an image of the photoacoustic signal based on the photoacoustic signal obtained from a wide range of the sample T by scanning the predetermined position of the sample T by the galvano scanner 29. To do. The image signal is sent to the display device of the illustration, and is stored in the storage device of the illustration as needed.

The control unit 45 controls the entire photoacoustic microscope 1. Specifically, it controls the emission timing of the optical pulse emitted from the pulse laser 10, the transmission control of the signal that triggers the function generator 31 to output the drive signal, the control of the drive unit of the galvano scanner 29, and the like. The control unit 45 also controls the photoacoustic detection unit 43 and the image processing unit 44.

In the above configuration, the observation device 50 according to the present embodiment includes a pulse laser 10, an irradiation optical system 20, and a detection unit 40. However, the scanning unit having the galvano scanner 29 and the objective lens 30 are not essential configurations for the observation device 50. Further, the irradiation optical system 20 includes a switching unit 51 for switching between the first mode and the second mode, and in the present embodiment, the switching unit 51 includes a first AOM 22, a second AOM 25, an optical fiber 24, and a function. A generator 31 is provided.

Next, with reference to FIG. 3, the operation and detection principle of the photoacoustic microscope 1 according to the present embodiment will be described.

FIG. 3A is a waveform diagram showing the pulsed light L emitted from the pulsed laser 10. The control unit 45 of the information processing apparatus 41 drives the pulse laser 10 so that the pulse L is emitted from the pulse laser 10 at a predetermined repeating frequency. As an example, the repetition frequency of the pulsed light L emitted from the pulse laser 10 is 80 MHz.

3 (b) and 3 (c) are waveform diagrams showing drive voltage signals supplied from the function generator 31 to the first AOM 22 and the second AOM 25, respectively. The drive voltage signal supplied from the function generator 31 is a rectangular wave having a frequency f, and its phase is inverted between the first AOM 22 and the second AOM 25. Therefore, the first AOM 22 and the second AOM 25 are alternately turned ON / OFF in the cycle of the frequency f as shown in the figure. The control unit 45 of the information processing device 41 sends out a trigger signal that causes the function generator 31 to output a drive voltage signal as shown in the figure.

FIG. 3D is a waveform diagram showing the optical LA output from the first AOM22, and FIG. 3E is a waveform diagram showing the optical LB output from the second AOM25. As shown in FIGS. 3 (d) and 3 (e), light LA and light LB are alternately emitted from the first AOM 22 and the second AOM 25.

The photoacoustic detection unit 43 detects a photoacoustic signal generated by irradiating the sample T with light LA or light LB. Then, the photoacoustic detection unit 43 calculates the intensity of the photoacoustic signal based only on the diphoton excitation by subtracting the intensity of the photoacoustic signal based on the optical LB from the intensity of the photoacoustic signal based on the optical LA. Therefore, the photoacoustic detection unit 43 or the control unit 45 has a schematic storage unit that temporarily stores photoacoustic signals based on the optical LA and LB.

The timing for calculating the intensity of the photoacoustic signal based only on two-photon excitation is arbitrary. However, in consideration of real-time performance, it is preferable to calculate at intervals equal to the frequency f of the drive voltage signal supplied from the function generator 31.

Next, the control unit 45 changes the irradiation positions of the light LA and the light LB incident on the sample T by controlling the galvano scanner 29. As an example, the irradiation position is changed from the spot SP1 shown in FIG. 1 to the spot SP2. Then, the photoacoustic detection unit 43 also performs the above calculation in the spot SP2.

By controlling the galvano scanner 29, the control unit 45 scans the optical LA and the optical LB on a predetermined plane of the sample T located at a predetermined depth in the incident direction of the optical LA and the optical LB. The photoacoustic detection unit 43 obtains the intensity of the photoacoustic signal based on the optical LB from the intensity of the photoacoustic signal based on the optical LA and the optical LB and the photoacoustic signal based on the optical LA at each scanning spot. The photoacoustic signal is calculated based only on the two-photon excitation. The image processing unit 44 is a distribution image of a photoacoustic signal based on optical LA and optical LB and a photoacoustic signal based only on two-photon excitation based on the photoacoustic signal calculated by the photoacoustic detection unit 43 at each scanning spot. To generate.

The position in the depth direction (Z direction) of the sample T irradiated with the light LA and the light LB is determined by adjusting the relative distance between the objective lens 30 and the sample T, and is fixed during image generation. .. The objective lens 30 may be moved or the sample T may be moved for the distance in the Z direction.

In the photoacoustic microscope 1 according to the present embodiment, the switching unit 51 has a first mode in which the sample T is generated with one-photon excitation and a two-photon excitation, and a second mode in which the sample T is generated with at least one photon excitation. I'm switching. Therefore, it is possible to acquire a photoacoustic signal based only on two-photon excitation by using a pulse laser 10 having a fixed pulse width as a light source. As a result, according to the photoacoustic microscope 1 according to the present embodiment, it is possible to measure the photoacoustic signal and acquire an image with a simple configuration. In addition, the work of changing the pulse width in the pulse laser 10 becomes unnecessary. As a result, the measurement of the photoacoustic signal and the acquisition of the image can be performed at high speed.

A fluorescence image can also be obtained by labeling the sample T with a fluorescent reagent and detecting the intensity of fluorescence emitted by the sample T with a photomultiplier tube 33.

(Second embodiment)
Next, with reference to FIG. 4, a photoacoustic microscope to which the observation device according to the second embodiment is applied will be described. FIG. 4 is a block diagram showing a schematic configuration of a photoacoustic microscope to which the observation device according to the second embodiment is applied. In the following description, the same components as those of the photoacoustic microscope 1 according to the first embodiment are designated by the same reference numerals, and the description thereof will be simplified.

The photoacoustic microscope 100 according to the present embodiment also has a pulse laser 10 as a light source, an irradiation optical system 110, and a detection unit 40, similarly to the photoacoustic microscope 1 according to the first embodiment.

The irradiation optical system 110 includes a spatial light modulator (Spatial Light Modulator, hereinafter referred to as SLM) 111, a mirror 112, a galvano scanner 29, and an objective lens 30.

The pulsed light L from the pulse laser 10 is incident on the SLM 111. The SLM111 is a device that modulates amplitude modulation, phase modulation, or polarization spatially and temporally. Since the SLM itself is a known device, a detailed description of its configuration will be omitted. As an example, the SLM 111 according to the present embodiment has a liquid crystal element whose refractive index can be controlled in pixel units. When the incident light L is incident on the liquid crystal element and reflected, phase modulation can be applied to the incident light L on a pixel-by-pixel basis. The control unit 45 of the information processing device 41 sends a signal for controlling the drive of the SLM 111. The SLM 111 is installed at a position conjugate with the pupil surface of the objective lens 30 by a lens (not shown).

The reflected light L from the SLM 111 is guided in a predetermined direction by the mirror 112 and is incident on the galvano scanner 29. Then, the light L is focused by the objective lens 30 via the galvano scanner 29, and is incident on the predetermined position of the sample T.

In the above configuration, the observation device 120 according to the present embodiment includes a pulse laser 10, an irradiation optical system 110, and a detection unit 40. However, the scanning unit having the galvano scanner 29 and the objective lens 30 are not essential configurations for the observation device 120. Further, the SLM 111 constitutes a switching unit included in the irradiation optical system 110.

Next, the operation and the detection principle of the photoacoustic microscope 100 according to the present embodiment will be described with reference to FIGS. 5 to 7.

The control unit 45 of the information processing apparatus 41 drives and controls the SLM 111 so as to give the reflected light L any of the optical profiles as shown in FIGS. 6 (a) and 6 (b) and 8 (a) and 8 (b). To do. Here, the upper part of FIG. 6 (a), (b), among the light L incident on the sample T, an optical profile of the defocus plane Z ₁ near the surface of the sample T shown in FIG. Further, the lower part of FIGS. 6A and 6B shows the focal plane Z at a predetermined depth in the Z direction from the surface of the sample T shown in FIG. 5, that is, the focal length of the light L incident on the sample T. ₂ optical profiles. 8 (a) and 8 (b) show the optical profiles shown in FIGS. 6 (a) and 6 (b) on the focal plane Z ₂ shown in FIG. 5, that is, the point spread function (hereinafter referred to as PSF). ) Is shown in the figure.

On the out-of-focus surface Z ₁ , the light L is not converged to one point (more precisely, a minute region), and is irradiated to a region having a predetermined width. Therefore, PSF is not formed by light L. Therefore, as shown in the upper part of FIGS. 6A and 6B, the optical profile has a uniform distribution.

On the other hand, the light L is focused on the focal plane Z ₂ . Therefore, the light L forms the PSF. The optical profile shown in the lower part of FIG. 6A and FIG. 8A, that is, the PSF has a peak at the focal position (the central position in FIG. 6 and the dotted line portion in the upper part of FIG. 8). On the other hand, the PSF shown in the lower part of FIG. 6B and FIG. 8B has a peak at a position other than the focal position. More specifically, it is a donut-shaped PSF having a peak on the circumference of a circle at a predetermined distance centered on the focal position. Therefore, the light L having the optical profile shown in FIGS. 6 (a) and 8 (a) is the light having the first intensity distribution on the focal plane Z ₂ , and is shown in FIGS. 6 (b) and 8 (b). The light L having the optical profile shown in is the light having the second intensity distribution on the focal plane Z ₂ .

The phase modulation provided by the SLM 111 for providing such a PSF will be described with reference to FIG. FIG. 7 is a diagram showing a phase distribution on the pupil surface of the objective lens 30. FIG. 7A is a phase distribution in the pupil function corresponding to the optical profile of FIG. 6A, which is a uniform and constant-valued phase. On the other hand, FIG. 7 (b) shows the phase distribution in the pupil function corresponding to the optical profile of FIG. 6 (b), which changes in the circumferential direction and changes from 0 to 2π when it goes around in the circumferential direction.

An optical instrument that gives phase modulation as shown in FIG. 7B is called a spiral phase plate (VPP). The SLM 111 is driven and controlled so as to give the reflected light L the phase distribution shown in FIG. 7A or FIG. 7B.

When the sample T is irradiated with the light L having the PSF shown in FIGS. 6 (a) and 8 (a) on the focal plane Z ₂ , the high-intensity light L is emitted at the focal position (dotted line portion in the upper part of FIG. 8). Be irradiated. As a result, photoacoustic signals based on one-photon excitation and two-photon excitation are generated at this focal position. This is the first mode. The photoacoustic wave W based on the generated photoacoustic signal is received by the transducer 42.

On the other hand, when the sample T is irradiated with the light L having the PSF shown in FIGS. 6 (b) and 8 (b) on the focal plane Z ₂ , the light L of low intensity is irradiated when considering only the focal position. (Dotted line in the lower part of FIG. 8). As a result, at this focal position, no photoacoustic signal based on two-photon excitation is generated, and a very small amount of photoacoustic signal based on one-photon excitation is generated.
On the other hand, since a peak is generated on the circumference of a circle at a predetermined distance centered on the focal position, a photoacoustic signal based on one-photon excitation and two-photon excitation is generated from this donut region. This is the second mode. The photoacoustic wave W based on the generated photoacoustic signal is received by the transducer 42.

Therefore, the control unit 45 of the information processing device 41 drives and controls the SLM 111 so as to alternately apply the PSFs shown in FIGS. 6A and 6B to the reflected light L. That is, the SLM 111 is a switching unit that switches between the first mode and the second mode. The PSF switching frequency by the SLM 111 is preferably high in consideration of real-time performance, but the switching frequency is determined in consideration of the calculation speeds of the image processing unit 44 and the control unit 45.

Alternatively, the control unit 45 applies a rectangular wave or a sine wave having a frequency f to the SLM 111, so that the SLM 111 controls PSF switching according to the frequency f. Further, the control unit 45 provides the lock-in signal of the frequency f to the photoacoustic detection unit 43 and the image processing unit 44, and performs various signal processing in the cycle of the frequency f.

At this time, the image I obtained from the photoacoustic signal based on the two-photon excitation is given by the following equation.
[Number 1]
I = I _{1 −} αI ₂
here,
I ₁ : Image acquired by PSF shown in FIG. 6 (a) I ₂ :: Image acquired by PSF shown in FIG. 6 (b) α: Proportional coefficient

In both the first mode and the second mode, a photoacoustic signal is generated by one-photon excitation on the out-of-focus surface Z ₁ and is mixed in each image. Therefore, by taking the difference between the two, it is possible to remove the photoacoustic signal based on the one-photon excitation generated from the out-of-focus surface Z ₁ and extract the photoacoustic signal based on the two-photon excitation generated from the focal plane Z _2. .. To remove the photoacoustic signal based on one-photon excitation resulting from defocus plane Z ₁ is, alpha = 1 is desirable. On the other hand, the intensity of the PSF peak in FIG. 6B and its vicinity in Image I becomes negative. Since the image I cannot take a negative value, information is lost in this part.

Therefore, it is desirable to make the value of α smaller than 1 in order to reduce this effect. However, in this case, the effect of photoacoustic signal removal based on one-photon excitation by subtraction is reduced. In this way, there is a trade-off depending on the value of α.

Therefore, at the time of generating the image I, after the image I ₁ and the image I ₂ are acquired, the α is changed from 0.5 to 1 to generate the image I. Then, it is desirable to select α in which the negative values of contrast and brightness are balanced in the image I. The magnitude of the contrast may be evaluated by the magnitude of the DC component of the Fourier transform of the image I. The negative luminance may be evaluated by the number of pixels taking a negative value or the sum of the luminance values of the pixels taking a negative value.

Since the difference in PSF occurs only in the focal plane Z ₂ , the photoacoustic signal generated from the out-of-focus plane Z ₁ can be removed by subtracting the pixel I ₂ from the image I ₁ . When the light intensity is increased, an undesired two-photon excitation photoacoustic signal may be generated even on the out-of-focus surface Z ₁ , but this signal can also be removed by this method.

Also in the photoacoustic microscope 100 according to the present embodiment, the first mode in which the sample T is generated with one-photon excitation and the two-photon excitation by the switching unit SLM111, and the second mode in which the sample T is generated with at least one photon excitation. Switching between modes. Therefore, it is possible to acquire a photoacoustic signal based only on two-photon excitation by using a pulse laser 10 having a fixed pulse width as a light source. As a result, the same effect as that of the photoacoustic microscope 1 according to the first embodiment described above can be obtained by the photoacoustic microscope 100 according to the present embodiment.

(Modification example)
Although the embodiment has been described in detail with reference to the drawings, the specific configuration is not limited to the embodiment and the embodiment, and design changes to the extent that the gist of the embodiment is not deviated are described in the present disclosure. included.

As an example, in the photoacoustic microscope 1 according to the first embodiment described above, a pulse laser 10 having a pulse width of about femtoseconds was used, but this pulse laser 10 and a pulse width of about picoseconds are provided. It may be used in combination with a pulse laser. More specifically, the pulsed light from the pulsed laser 10 may be directly incident on the first AOM 22, and the pulsed light from the pulsed laser having a pulse width of about picoseconds may be directly incident on the second AOM 25. In this case, the half mirror 21 can be omitted.

Further, in the first embodiment, an example in which the optical fiber 24 which is the pulse width extension portion is arranged only in the optical path in the second mode is shown, but in addition to this, the pulse extension portion is also shown in the optical path in the first mode. May be placed. In this case, the amount of extension by the pulse extension part of the second mode is made larger than the amount of extension by the pulse extension part of the first mode. As a result, even if the pulse width of the light from the light source 10 is smaller than the pulse width suitable for generating two-photon excitation at the focal plane in the first mode, the light emitted to the sample T in the first mode The pulse width can be set to an appropriate size.

Further, in the photomultiplier microscope 100 according to the second embodiment, similarly to the photomultiplier microscope 1 according to the first embodiment, a dichroic mirror 28 is inserted between the mirror 112 and the galvano scanner 29 to perform optical optics. A filter 32 and a photomultiplier tube 33 may be added.

As a result, a fluorescence image can be obtained by labeling the sample T with a fluorescent reagent and detecting the intensity of fluorescence emitted by the sample T with the photomultiplier tube 33.

Then, in the above-mentioned first and second embodiments, the observation device is applied to the photoacoustic microscope, but the observation device can also be applied to other devices. As an example, the observation device can also be applied to an endoscopic device.

The endoscopic device to which the observation device according to the first and second embodiments is applied includes an insertion portion having a tube member inserted into the body cavity of the subject and an operation unit that operates the tip portion of the insertion portion. And have. The observation device according to the first to second embodiments is arranged at the tip of the insertion portion. Even with an endoscope device having such a configuration, it is possible to acquire a photoacoustic signal based only on two-photon excitation generated from a sample T such as an inner surface of a mucous membrane in a subject. In addition, if a scanning unit is provided, it is possible to acquire a photoacoustic signal image based only on two-photon excitation of the inner surface of the mucous membrane in the subject.

As a sequence for acquiring an opto-acoustic image, a configuration for switching between the first mode and the second mode for each pixel of the image or for each predetermined number of pixels, and image acquisition in the first mode (acquisition of signals for all pixels). Then, there are two types of configurations: image acquisition (acquisition of signals for all pixels) in the second mode. The former is more preferable because it is less affected by the movement of the sample and the fluctuation of the device.

L light LA light LB light T sample 1,100 Photoacoustic microscope 10 Pulsed laser (light source)
20, 110 Irradiation optical system 22 First AOM
24 Optical fiber (pulse width extension part)
25 Second AOM
29 Galvano scanner (scanning unit)
31 Function generator 40 Detection unit 44 Image processing unit (Image generation unit)
51 Switching unit 111 SLM (switching unit)

Claims (11)

A light source that generates light and
An irradiation optical system that irradiates the sample with the light,
It has a detection unit that detects a signal associated with the reaction of the sample due to the irradiation of the light.
The irradiation optical system has a first mode in which one-photon excitation and two-photon excitation are generated in the sample by irradiating light having a first intensity distribution on the focal plane, and a second intensity distribution on the focal plane. An observation device characterized by having a switching unit that switches between a second mode in which at least one photon excitation is generated in the sample by irradiating the sample with the light. The observation device according to claim 1, wherein the switching unit switches between the first mode and the second mode at a predetermined timing. The first or second aspect of the present invention, wherein the detection unit detects a difference between the signals by subtracting the signal detected by the second mode from the signal detected by the first mode. Observation device. The third aspect of claim 3, wherein the detection unit detects a signal based on two-photon excitation by subtracting the signal detected by the second mode from the signal detected by the first mode. Observation device. In the first mode, the switching unit irradiates the sample with the light having a predetermined pulse width, and in the second mode, irradiates the sample with light having a pulse width longer than the pulse width. The observation device according to any one of claims 1 to 4. The switching unit has a pulse width extending portion that expands the pulse width of the light generated from the light source, and the pulse width extending portion expands the pulse width of the light at least in the second mode. The observation device according to claim 5. In the first mode, the switching unit irradiates the focal position of the focal plane at a specific depth of the sample with the light, and in the second mode, the switching portion is located at a position other than the focal position on the focal plane. The observation device according to any one of claims 1 to 3, further comprising irradiating light. The switching unit has a point image distribution function at the focal position of the light irradiating the sample in the first mode and a point at the focal position of the light irradiating the sample in the second mode. The observation device according to claim 6, wherein the image distribution function is different from the image distribution function. By irradiating the sample with light having a first intensity distribution on the focal plane to generate one-photon excitation and two-photon excitation, and by irradiating the sample with light having a second intensity distribution. A step of switching between the second mode in which at least one photon excitation is generated in the sample, and
A step of detecting a signal accompanying the reaction of the sample due to the irradiation of the light, and
An observation method comprising. The observation device according to any one of claims 1 to 8.
A microscope device including an image generation unit that generates an image based on the signals corresponding to a plurality of positions of the sample. An insertion part having a tube member to be inserted into the body cavity of the subject,
It has an operation part that operates the tip part of the insertion part, and has an operation part.
An endoscopic apparatus according to any one of claims 1 to 8, wherein the observation apparatus is arranged at the tip end portion of the insertion portion.