WO2015182269A1 - Image acquisition device and image acquisition method - Google Patents

Abstract

[Problem] To make a device more compact and more stably acquire a fluorescence image through a multiphoton excitation process. [Solution] An image acquisition device according to the present disclosure is provided with: a light-source optical system for guiding, to an imaging subject, excitation light for exciting and generating fluorescence in the imaging subject using two or more photons; image guide fiber in which a plurality of multimode optical fibers are bundled and that transmits excitation light having entered one end thereof to the imaging subject and transmits, to the one end, an image of the imaging subject formed at the other end as a result of the fluorescence generated in the imaging subject; an imaging optical system for scanning the image of the imaging subject at a scanning pitch that is narrower than the size of the cores of the plurality of optical fibers, imaging so that at least portions of areas corresponding to the optical fibers are included in a plurality of images, and generating a plurality of image data items for the imaging subject; and a selection unit for selecting, for each of the plurality of pixels composing the areas corresponding to the optical fibers, the pixel value having the highest brightness from among the plurality of image data items as a representative pixel value.

Description

Image acquisition apparatus and image acquisition method

The present disclosure relates to an image acquisition device and an image acquisition method.

As a laser confocal microscope (microscope endoscope) system using an image guide fiber composed of a plurality of optical fiber strands, for example, those shown in the following Patent Document 1 and Non-Patent Document 1 have been proposed. .

For example, a microscopic endoscope system proposed in Patent Document 1 transmits fluorescence generated by exciting an observation target with one photon through an image guide fiber, and observes the generated fluorescence. Further, for example, the microscopic endoscope system proposed in Non-Patent Document 1 transmits fluorescence generated by exciting an observation target with two photons using a GRIN lens and an image guide fiber, and the generated fluorescence is transmitted. To observe.

These microscopic endoscope systems can observe an observation object that cannot be accessed with a conventional microscope because the image guide fiber has a thin diameter of about 1 mm.

JP-T-2007-530197

W. Gobel, J. et al. N. D. Kerr, A .; Nimmerjahn, F.M. Helmchen, “Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective”, OPTICS LETTER 200. 29, no. 21, p. 2521.

However, as will be described in detail below, the microscopic endoscope system proposed in Patent Document 1 uses an image guide fiber made of a multi-mode optical fiber, so that two-photon excitation is performed as it is. It cannot be applied to a fluorescence microscope system using the process.

The microscopic endoscope system proposed in Non-Patent Document 1 is said to use a two-photon excitation process. In such a microscopic endoscope system, a blur filter such as a Gaussian filter is applied to the obtained image without considering the order of the mode in which the optical fiber constituting the image guide fiber is guided. As will be described in detail below, the fluorescence efficiency of the fluorescence due to the two-photon excitation process deteriorates, and the fluorescence signal cannot be acquired stably.

Therefore, in view of the above circumstances, the present disclosure proposes an image acquisition device and an image acquisition method capable of acquiring a fluorescence image by a multiphoton excitation process more stably while reducing the size of the device. .

According to the present disclosure, a light source optical system that guides excitation light for generating fluorescence by exciting the imaging target with two or more photons to the imaging target, and a multimode optical fiber Are bundled, and the excitation light incident on one end by the light source optical system is transmitted to the object to be imaged and to the other end by the fluorescence generated by the object to be imaged. An image guide fiber that transmits the image of the imaged object that is imaged to the one end, and an image of the imaged object that is transmitted to the one end of the image guide fiber Scanning at a scanning pitch narrower than the core size of the plurality of optical fiber strands so that at least a part of the corresponding region of the optical fiber corresponding to each of the optical fiber strands is included in the plurality of images. Imaged in the For each of the imaging optical system that generates a plurality of image data of the imaging body and the plurality of pixels that constitute the region corresponding to the optical fiber, the pixel value that has the maximum luminance among the plurality of the image data is determined for the pixel. An image acquisition device is provided that includes a selection unit that selects a representative pixel value.

In addition, according to the present disclosure, excitation light for generating fluorescence by exciting the imaging target with two or more photons is guided to the imaging target, and a multimode optical fiber The excitation light incident on one end of the image guide fiber is transmitted to the object to be imaged by a plurality of bundled image guide fibers, and the other end by the fluorescence generated in the object to be imaged Transmitting the image of the object to be imaged to the one end, and transmitting the image of the object to be transmitted to the one end of the image guide fiber to the one end. Scanning at a scanning pitch narrower than the core size of the plurality of optical fiber strands so that at least a part of the corresponding region of the optical fiber corresponding to each of the optical fiber strands is included in the plurality of images. Imaging Generating a plurality of image data of the object to be imaged and, for each of a plurality of pixels constituting the corresponding region of the optical fiber, a pixel value having a maximum brightness among the plurality of the image data And selecting as a representative pixel value of the image.

According to the present disclosure, when an imaging target is irradiated with excitation light having a predetermined wavelength to excite the imaging target with two or more photons, fluorescence is generated and a plurality of multimode optical fiber strands are bundled together. The image of the imaged object formed on one end of the image guide fiber is transmitted to the other end of the image guide fiber, and the transmitted image of the imaged object is converted into a plurality of light beams. Image data of the object to be imaged by scanning at a scanning pitch narrower than the size of the core of the fiber strand and imaging so that at least a part of the corresponding region of each optical fiber strand is included in a plurality of images Are generated, and for each of a plurality of pixels constituting the corresponding region of the optical fiber, a pixel value having the maximum luminance among the plurality of image data is selected as a representative pixel value of the pixel.

As described above, according to the present disclosure, it is possible to more stably acquire a fluorescence image by a multiphoton excitation process while downsizing the apparatus.

Note that the above effects are not necessarily limited, and any of the effects shown in the present specification or other things that can be grasped from the present specification together with the above effects or instead of the above effects. The effect of may be produced.

It is explanatory drawing for demonstrating the mode of the light excited by the optical fiber strand. It is explanatory drawing for demonstrating the difference in the arrival time of the light which guides an optical fiber strand. It is explanatory drawing for demonstrating the relationship between the mode of the light which guides an optical fiber strand, and the brightness | luminance of the fluorescence to generate | occur | produce. FIG. 3 is an explanatory diagram schematically illustrating a configuration of an image acquisition device according to a first embodiment of the present disclosure. It is explanatory drawing which showed typically the structure of the microscope unit with which the image acquisition apparatus which concerns on the same embodiment is provided. It is explanatory drawing which showed typically an example of the light source with which the microscope unit which concerns on the same embodiment is provided. It is explanatory drawing which showed typically an example of the light source with which the microscope unit which concerns on the same embodiment is provided. It is explanatory drawing which showed typically an example of the light source with which the microscope unit which concerns on the same embodiment is provided. It is explanatory drawing which showed typically an example of the light source with which the microscope unit which concerns on the same embodiment is provided. It is explanatory drawing which showed typically the structure of the image guide fiber with which the microscope unit which concerns on the same embodiment is provided. It is explanatory drawing which showed typically the scanning method of the image guide fiber in the microscope unit which concerns on the embodiment. It is explanatory drawing which showed typically the scanning method of the image guide fiber in the microscope unit which concerns on the embodiment. It is the block diagram which showed typically the structure of the arithmetic processing unit with which the image acquisition apparatus which concerns on the same embodiment is provided. It is explanatory drawing for demonstrating the selection process of the representative pixel value in the arithmetic processing unit which concerns on the embodiment. It is explanatory drawing for demonstrating the selection process of the representative pixel value in the arithmetic processing unit which concerns on the embodiment. It is explanatory drawing for demonstrating the selection process of the representative pixel value in the arithmetic processing unit which concerns on the embodiment. It is the flowchart which showed an example of the flow of the image acquisition method which concerns on the embodiment. It is the block diagram which showed an example of the hardware constitutions of the arithmetic processing unit which concerns on the same embodiment. It is explanatory drawing which showed typically the structure of the microscope unit used in the experiment example. It is explanatory drawing which showed the fluorescence image imaged in the experiment example. It is explanatory drawing which showed the fluorescence image imaged in the experiment example. It is explanatory drawing which showed the fluorescence image imaged in the experiment example. It is explanatory drawing which showed the fluorescence image imaged in the experiment example. It is explanatory drawing which showed the fluorescence image imaged in the experiment example. It is explanatory drawing which showed the fluorescence image imaged in the experiment example.

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In addition, in this specification and drawing, about the component which has the substantially same function structure, duplication description is abbreviate | omitted by attaching | subjecting the same code | symbol.

The description will be made in the following order.
1. 1. Study by the present inventor First embodiment 2.1. Configuration of image acquisition device 2.2. 2. Image acquisition method 3. Hardware configuration of arithmetic processing unit Experimental example

(About examination by the present inventor)
Prior to the description of the image acquisition device according to the embodiment of the present disclosure, a result of a study on an image acquisition device capable of acquiring fluorescence by the multiphoton excitation process performed by the present inventor will be briefly described.

In the laser microscope endoscope as disclosed in Patent Document 1 and Non-Patent Document 1, the image guide fiber is focused on the end surface on the main body side (light source or detector side) with a galvanometer mirror used in the laser microscope. A scanning method is used. As a result, the scan spot at the end surface of the image guide fiber on the main body side is projected onto the end surface on the sample side, and information from the sample is acquired by the spot projected onto the end surface on the sample side. That is, when the laser microscope to be used is a fluorescence microscope, the sample is fluorescently excited by the projected spot light, and the fluorescence emitted from the sample is guided again to the image guide fiber, thereby receiving the light received on the main body side. The fluorescent light generated in the element is guided.

Here, a general image guide fiber is a bundle of a plurality of optical fiber strands. In such an image guide fiber, in order to make the core, which is an optical waveguide, close to each other with high density, the refractive index difference between the core and the clad of the optical fiber is increased, and the optical fiber element is used to achieve uniform image sensitivity. It is important to make the optical fiber used in the line multi-mode, and to reduce the width at which the electric field intensity distribution in the clad portion exudes in order to prevent crosstalk.

FIG. 1 is an explanatory diagram for explaining a mode of light excited by an optical fiber, and schematically shows an enlarged part of the optical fiber constituting the image guide fiber. . In a general image guide fiber, when a plurality of optical fiber strands are bundled (bundled), the optical fiber strands are arranged so that each optical fiber strand has a hexagonal fine structure as much as possible. However, looking at the cross-sectional structure of the manufactured optical fiber, as shown schematically in FIG. 1, the cores C _A to C _E of the optical fiber have a substantially hexagonal fine structure. However, the array is distorted.

Here, as the core C _A, if the excitation light emitted from the light source is irradiated on the entire core to include a central portion of the core C _A, the core C _A, mainly the fundamental (zero-order mode Light: light in a mode in which the intensity of the central portion is increased in the intensity distribution of the core cross section. Also, if the excitation light is irradiated so as to extend to the core C _B and the core C _C, the core C _B excitation light is irradiated to a region of a large portion including the central portion of the core, and mainly the fundamental primary Waves (light in the first-order mode: light in a mode in which two antinodes exist in the intensity distribution of the core cross section) are excited, while excitation light is irradiated to a part of the region that does not include the center of the core in the core C _C, mainly primary wave is excited. In addition, when the excitation light is irradiated only to a part of the region that does not include the central part of the core, such as the core _CD and the core _CE , in each core, the primary wave or higher order is mainly generated. The mode is excited.

By the way, the intensity (signal amount) of the fluorescence generated by the one-photon excitation process is proportional to the excitation light energy, while the intensity (signal amount) of the fluorescence generated by the two-photon excitation process is the square of the excitation light energy. It is known to be proportional.

Since the microscopic endoscope system disclosed in Patent Document 1 is a system using a one-photon excitation process, the first-order mode is used regardless of whether the mode of light guided through the optical fiber is the zero-order mode. Regardless of the mode, information (that is, a fluorescence signal) can be acquired according to the sum of the light energy at the sample side end face, regardless of the mode. Therefore, in a system using a one-photon excitation process as disclosed in Patent Document 1, the size of the focused spot on the end face of the image guide fiber is slightly larger than the core diameter of the optical fiber. As a result, a stable fluorescent image can be acquired.

One of the standard image guide fiber configurations is an image guide fiber having the following set values.
Core diameter = about 3 μm
Refractive index of _core n _core = 1.4903276
Refractive index of _clad n _clad = 1.42324508
Wavelength of guided light λ = 1300nm

When the present inventor conducted the mode calculation of the optical fiber in the configuration of the image guide fiber, the effective refractive index of the 0th-order mode (fundamental wave) becomes 1.4702, and the first-order mode (first-order wave). The effective refractive index of was 1.4406. As schematically shown in FIG. 2, when the length of the image guide fiber is 2 m, the light of the first mode is 198 picoseconds faster than the zeroth mode on the sample side end face. Indicates that it will reach.

In the system of Patent Document 1 using a one-photon excitation process, even if there is a difference of 198 picoseconds in the arrival times of light in the 0th order mode and the 1st order mode, the amount of fluorescence proportional to the energy of each light can be obtained. Therefore, the total amount of fluorescence does not change. However, as described above, in the case of a system using a two-photon excitation process, the amount of fluorescence signal obtained is proportional to the square of the energy of the excitation light, so that the light guided through the optical fiber is 0. If the light is separated into the next mode light and the first mode light, the amount of fluorescence signal generated by the two-photon excitation process is reduced.

FIG. 3 is an explanatory diagram for explaining the relationship between the mode of light guided through the optical fiber and the luminance of the generated fluorescence. The light guided through the optical fiber is irradiated onto the fluorescent beads. The brightness of the fluorescent light generated in this case is roughly classified into patterns A to E.

Consider the case where the 0-order mode excitation light is irradiated to the fluorescent beads located at the center of the core as shown in Pattern A. In this case, the luminance of the generated fluorescence is large (that is, bright fluorescence is generated), whether it is fluorescence by a one-photon excitation process or fluorescence by a two-photon excitation process. In addition, as shown in pattern B, when the excitation light in the first-order mode is irradiated onto the fluorescent bead located at the center of the core, the fluorescence intensity by the one-photon excitation process is high, whereas the two-photon excitation is performed. The brightness of the fluorescence due to the process is not very large.

In addition, as shown in the pattern C and the pattern D, when the fluorescent beads are not located at the center of the core, the luminance of the fluorescence due to the one-photon excitation process is increased regardless of the mode of the excitation light. The luminance of the fluorescence due to the two-photon excitation process does not increase so much regardless of the mode of excitation light.

In addition, as shown in the pattern E, when the fluorescent beads are positioned away from the core, the luminance of the generated fluorescence becomes small regardless of the excitation process.

As is clear from FIG. 3, in order to increase the luminance of fluorescence by the two-photon excitation process, it is preferable that the excitation light is guided in a single mode (more specifically, the 0th order mode). Further, just using an image guide fiber composed of a multimode optical fiber as in Patent Document 1 causes a decrease in fluorescence luminance and a variation in fluorescence luminance, resulting in a two-photon excitation process (and further It can also be seen that it is not possible to acquire a fluorescence image using a multiphoton excitation process).

When using an image guide fiber to measure fluorescence due to a multiphoton excitation process, it is possible to use a single-mode optical fiber as the optical fiber that constitutes the image guide fiber. In this case, in order to reduce crosstalk between adjacent optical fiber strands when using a single mode optical fiber strand, it is necessary to reduce the difference in refractive index between the core and the clad of the optical fiber. However, reducing the refractive index difference between the core and the cladding means increasing the oozing of the electric field strength distribution to the cladding. Accordingly, in order to reduce crosstalk between adjacent optical fiber strands, it is important to widen the distance between adjacent optical fiber strands.

Here, the resolution of the image guide fiber depends on the arrangement interval of the optical fiber, and the smaller the arrangement interval of the optical fiber, the higher the obtained resolution. Therefore, when a single mode optical fiber is used and the interval between the optical fibers is widened, the optical fiber is scanned to obtain the same resolution as that of a normal image guide fiber. While a further mechanism is required, it is difficult to reduce the diameter.

Furthermore, reducing the refractive index difference between the core and the clad of the optical fiber lowers the numerical aperture (NA) of the optical fiber, so that the fluorescence signal by the two-photon excitation process is efficiently acquired. For this, it is important to use a double clad fiber.

As described above, when an image acquisition method similar to that of Patent Document 1 is used using an image guide fiber composed of a multimode optical fiber, a fluorescent image obtained by the two-photon excitation process is stabilized. It cannot be acquired. In addition, in order to perform the two-photon excitation process stably, when the optical fiber is composed of a single mode optical waveguide, the resolution of the obtained image is lowered, so that the diameter is reduced (that is, the size is reduced). Will become difficult.

Further, the microscopic endoscope system proposed in Non-Patent Document 1 does not consider the order of the mode for guiding the optical fiber constituting the image guide fiber, and is the same as that in Patent Document 1 described above. A fluorescence image is acquired by an image acquisition method, and a blur filter such as a Gaussian filter is applied. For this reason, even when the system proposed in Non-Patent Document 1 is used, it is not possible to stably obtain fluorescence due to the two-photon excitation process.

Based on the above knowledge, the present inventor is an image acquisition apparatus capable of stably acquiring a multiphoton excitation fluorescence image even when an image guide fiber made of a multimode optical fiber is used. , Earnestly examined. As a result, (1) if the single mode is excited at the incident end face of the image guide fiber, there may be a case where the sample side end face is reached in the single mode stochastically. The image acquisition device and the image acquisition method according to the embodiment of the present disclosure as described below are conceived that it is possible to acquire the data of the excitation light that has reached the sample side end surface in the single mode by selecting a high fluorescence value. Was completed.

(First embodiment)
Hereinafter, the image acquisition device according to the first embodiment of the present disclosure, which has been completed based on the above knowledge, will be described in detail with reference to FIGS.

<About image acquisition device>
[Overall configuration of image acquisition device]
First, the overall configuration of the image acquisition apparatus 1 according to the present embodiment will be described with reference to FIG. FIG. 4 is an explanatory diagram schematically showing the configuration of the image acquisition apparatus according to the present embodiment.

The image acquisition device 1 according to the present embodiment irradiates an imaging target that is an imaging target with excitation light having a predetermined wavelength, generates fluorescence by a multi-photon excitation process from the imaging target, and performs imaging based on the fluorescence. This is an apparatus for acquiring a captured image of an imaging body. As shown in FIG. 4, the image acquisition apparatus 1 includes a microscope unit 10 and an arithmetic processing unit 20.

The microscope unit 10 is a unit that generates image data relating to the generated fluorescence by irradiating the imaging target with excitation light having a predetermined wavelength and detecting the fluorescence generated by the multiphoton excitation process. Image data generated by the microscope unit 10 is output to the arithmetic processing unit 20. The detailed configuration of the microscope unit 10 will be described in detail below.

The arithmetic processing unit 20 comprehensively controls the imaging processing of the imaging target by the microscope unit 10 and performs arithmetic processing to be described later on the image data generated by the microscope unit 10 to obtain a captured image of the imaging target. It is a unit to generate.

The arithmetic processing unit 20 may be an information processing device such as various computers or servers provided outside the microscope unit 10, or may be a CPU (Central Processing Unit), ROM ( An arithmetic chip including a read only memory (RAM), a RAM (random access memory), or the like may be used.

The detailed configuration of the arithmetic processing unit 20 will be described in detail below.

[Configuration of Microscope Unit 10]
Next, a detailed configuration of the microscope unit 10 according to the present embodiment will be described with reference to FIGS. 5 to 8B.
FIG. 5 is an explanatory diagram schematically showing a configuration of a microscope unit included in the image acquisition device 1 according to the present embodiment. 6A to 6D are explanatory views schematically showing an example of a light source provided in the microscope unit according to the present embodiment. FIG. 7 is an explanatory view schematically showing the structure of the image guide fiber provided in the microscope unit according to the present embodiment. 8A and 8B are explanatory views schematically showing a scanning method of the image guide fiber in the microscope unit according to the present embodiment.

The microscope unit 10 included in the image acquisition apparatus 1 according to the present embodiment mainly includes a light source optical system 101, an image guide fiber 103, and an imaging optical system 105.

The light source optical system 101 guides excitation light having a predetermined wavelength for generating fluorescence by exciting the imaging target S with two or more photons (ie, exciting with multi-photons) to the imaging target S. It is an optical system. The light source optical system 101 includes a laser light source that emits excitation light having a predetermined wavelength, optical elements such as various lenses, various mirrors, and various filters that guide the excitation light emitted from the light source to the imaging target S. Consists of

The detailed arrangement of various optical elements in the light source optical system 101 is not particularly limited, and a known optical system can be employed.

Also, the laser light source included in the light source optical system 101 is not particularly limited, and various light sources such as various semiconductor lasers, solid-state lasers, and gas lasers can be used. By using a light source using various semiconductor lasers as the laser light source, the image acquisition device 1 can be further downsized.

As a light source using a semiconductor laser that can be used as a laser light source unit included in the light source optical system 101, for example, a semiconductor laser as shown in FIGS. 6A to 6D and a wavelength conversion unit that converts the wavelength of the semiconductor laser light are used. A light source can be used.

FIG. 6A schematically shows a main oscillator 111 composed of a semiconductor laser and a resonator, which is an example of a semiconductor laser applicable to a laser light source unit. The main oscillator 111 provided as a laser light source unit includes a semiconductor laser unit 112 capable of emitting laser light having a predetermined wavelength (for example, wavelength 405 nm), and a resonator unit for amplifying the laser light emitted from the semiconductor laser unit 112. 113.

FIG. 6B schematically shows a master oscillator output amplifier (MOPA) 114 composed of a master oscillator and an optical amplifier, which is an example of a semiconductor laser applicable to a laser light source unit. is there. In such a light source, an optical amplifier 115 for further amplifying the emitted laser light is provided after the main oscillator 111 shown in FIG. 6A. As the optical amplifier 115, for example, a semiconductor optical amplifier (SOA) or the like can be suitably used.

FIG. 6C schematically shows a light source having a MOPA 114, an optical amplifier, and a wavelength conversion unit, which is an example of a light source using a wavelength conversion unit that converts the wavelength of the semiconductor laser light applicable to the laser light source unit. Is. In such a light source, a wavelength converter 116 for converting the wavelength of the laser light whose intensity has been amplified is provided after the optical amplifier 115 shown in FIG. 6B. As the wavelength converter 116, for example, an optical parametric oscillator (Optical Parametric Oscillator: OPO) using various nonlinear crystals can be suitably used. Further, as shown in FIG. 6D, a beam shape correction unit 117 for correcting the beam shape of the laser light is provided between the MOPA 114 and the wavelength conversion unit 116, and the wavelength conversion efficiency in the wavelength conversion unit 116 is further improved. Also good.

Further, the wavelength of the excitation light emitted from the laser light source is not particularly limited, and a wavelength suitable for exciting the phosphor included in the imaging target S may be appropriately selected. Furthermore, the laser used as the light source may be a CW (Continuous Wave) laser or a pulsed laser.

The image guide fiber 103 is a bundle of a plurality of multi-mode optical fiber wires 121 as schematically shown in FIG. Each optical fiber 121 is formed of a core 123 capable of guiding not only 0th-order mode light but also higher-order mode light, and a clad 125 provided so as to cover the core 123. ing. Each optical fiber 121 is arranged so as to have a hexagonal fine structure as much as possible, as schematically shown in FIG. The separation distance d between the adjacent optical fiber wires 121 may be set as appropriate according to the required image resolution, and may be set to a value such as 3.5 μm, for example. Further, the diameter d ′ of the core 123 may be set as appropriate, for example, a value such as 3 μm.

The image guide fiber 103 transmits the excitation light incident on one end (for example, the end A in FIG. 5) by the light source optical system 101 to the imaging target S, and by the fluorescence generated in the imaging target S. An image of the imaging target S that is focused on the other end (for example, end B in FIG. 5) is transmitted to the other end (for example, end A in FIG. 5).

The imaging optical system 105 uses the size of the core 123 of the plurality of optical fiber strands 121 (see FIG. 7) as the image of the imaging target transmitted to the end of the image guide fiber 103 (for example, the end A in FIG. 5). Scanning at a scanning pitch narrower than the core diameter d ′). At this time, the imaging optical system 105 performs imaging so that at least a part of the corresponding region of the optical fiber corresponding to each optical fiber 121 is included in a plurality of images, and image data of the imaging target S is obtained. Generate multiple.

The imaging optical system 105 includes various detectors that detect an image of the imaging target transmitted through the image guide fiber 103 (that is, a fluorescent signal corresponding to the generated fluorescence), and the imaging target to the detector. It comprises optical elements such as various lenses, various mirrors, and various filters that guide an image (fluorescence signal).

The detailed arrangement of various optical elements in the imaging optical system 105 is not particularly limited, and a known optical system can be employed.

As the detector provided in the imaging optical system 105, a known one can be used as long as it can convert information on the intensity of fluorescence into an electrical signal. Examples of such detectors include various detectors such as a CCD (Charge-Coupled Device) and a photomultiplier tube (PMT).

Further, at least one of the light source optical system 101 and the imaging optical system 105 shown in FIG. 5 is provided with a mechanism for scanning the end face of the image guide fiber 103 as described in detail below. Such a scanning mechanism is not particularly limited, and for example, a known mechanism such as a galvanometer mirror can be used.

The configuration of the microscope unit 10 according to the present embodiment has been described in detail above with reference to FIGS.

[Image guide fiber scanning method]
Next, a method for scanning the end face of the image guide fiber 103 by the imaging optical system 105 will be described in detail with reference to FIGS. 8A and 8B.

As described above, the present inventor has found that if a single mode (0th-order mode) is excited at the incident end face of the image guide fiber, there may be a case where the specimen side end face may reach the sample side end face in a single mode. As a result, a method for scanning the end face of the image guide fiber 103 as schematically shown in FIGS. 8A and 8B was conceived.

That is, the microscope unit 10 according to the present embodiment scans the end surface of the image guide fiber 103 (for example, the end surface of the end A in FIG. 5) at a scanning pitch narrower than the core size of the plurality of optical fiber strands.

For example, FIG. 8A shows an example in which scanning is performed in a direction parallel to the scanning direction of the end face of the image guide fiber 103 at a pitch p narrower than the core size d ′ of the optical fiber strand 121. In such a case, the position of the axis representing the scanning direction (scanning axis) is set in advance according to the diameter of the image guide fiber 103 to be used, the core diameter d ′ of the optical fiber 121, and the like. Under the control, the end face of the image guide fiber 103 is imaged at the position of the black circle in FIG. 8A. At this time, the imaging interval in the direction parallel to the scanning direction is controlled by the arithmetic processing unit 20 based on the scanning pitch p, but the imaging interval in the direction orthogonal to the scanning direction is Control is based on the distance d between adjacent optical fiber strands 121. In the scanning method illustrated in FIG. 8A, the entire imaging target S is imaged once by the control described above. That is, in the scanning method shown in FIG. 8A, the frequency of image data generated by imaging (data restoration frequency) is higher than the number of optical fiber wires 121.

For example, FIG. 8B shows an example in which scanning is performed at a pitch p narrower than the core size d ′ of the optical fiber 121 in the direction orthogonal to the scanning direction of the end face of the image guide fiber 103. Yes. In such a case, the position of the axis representing the scanning direction (scanning axis) is set in advance according to the diameter of the image guide fiber 103 to be used, the core diameter d ′ of the optical fiber 121, and the like. Under control, the end face of the image guide fiber 103 is imaged at the position of the black circle in FIG. 8B. At this time, the imaging interval in the direction parallel to the scanning direction is controlled by the arithmetic processing unit 20 based on the interval d between the adjacent optical fiber strands 121 of the image guide fiber 103 and is orthogonal to the scanning direction. The imaging interval is controlled based on the scanning pitch p. In the scanning method shown in FIG. 8B, the whole image pickup object S is imaged a plurality of times (for example, 5 times in the example of FIG. 8B) by the control described above. That is, in the scanning method shown in FIG. 8B, the data restoration frequency in one scanning corresponds to the number of optical fiber strands 121, and the reference position (scanning start position) in each scanning process is the optical fiber element. It changes at a pitch p narrower than the arrangement pitch of the lines 121.

The “imaging” in the above description means that the excitation light guided by the light source optical system 101 forms an image at each black circle position in FIGS. 8A and 8B, and the image guide fiber 103 at the black circle position. This means that an image (fluorescent image) transmitted to the end face of the camera is taken.

By realizing the scanning method as described above, at least a part of a region corresponding to each optical fiber 121 (hereinafter also referred to as “optical fiber corresponding region”) is formed into a plurality of images. An image is taken so as to be contained.

By scanning the end face of the image guide fiber 103, as shown in FIGS. 8A and 8B, in the plurality of image data generated by the imaging processing, for example, as shown in the core C _A in FIG. 1, the excitation Image data in a situation where light is irradiated to the entire core is included. In situations such as those shown in the core C _A in FIG. 1, since the fundamental wave in the core of the image guide fiber 103 (0-order mode) is excited, stochastically also occur when reaching the fundamental to the sample end surface It becomes.

Note that at each imaging position as shown in FIGS. 8A and 8B, the microscope unit 10 may perform the imaging process only once, and in order to further increase the probability, imaging at a plurality of times at each imaging position. Processing may be performed.

Further, in the microscope unit 10 according to the present embodiment, a scanning method in which FIGS. 8A and 8B are combined (that is, a method of scanning at the scanning pitch p in each of the scanning direction and the direction orthogonal to the scanning direction). It goes without saying that may be adopted.

The specific size of the scanning pitch p shown in FIGS. 8A and 8B may be set as appropriate according to the core diameter d ′ of the optical fiber 121, but is about 1/10 of the core diameter d ′. It is preferable. As a result of the study by the present inventor, the scanning pitch p is set to 1/10 or less of the core diameter d ′ (for example, when the core diameter d ′ is 3 μm, the scanning pitch p is set to 0.3 μm or less). A luminance of 86% or more with respect to the maximum luminance excited by the strand 121 can be obtained. Also, by setting the scanning pitch p to 1/12 or less of the core diameter d ′, it is possible to obtain a luminance of 90% or more with respect to the maximum luminance excited by the optical fiber 121.

It should be noted that the scanning direction and imaging position shown in FIGS. 8A and 8B and the number of scans shown in FIG. 8B are merely examples, and are not limited to the examples shown in FIGS. 8A and 8B.

The example of the method for scanning the end face of the image guide fiber 103 according to the present embodiment has been specifically described above with reference to FIGS. 8A and 8B.

[Configuration of the arithmetic processing unit 20]
Next, the configuration of the arithmetic processing unit 20 included in the image acquisition device 1 according to the present embodiment will be described in detail with reference to FIGS. 9 to 12.
FIG. 9 is a block diagram schematically showing the configuration of the arithmetic processing unit provided in the image acquisition apparatus according to the present embodiment. FIG. 10 to FIG. 12 are explanatory diagrams for explaining representative pixel value selection processing in the arithmetic processing unit according to the present embodiment.

The arithmetic processing unit 20 according to the present embodiment controls the operation of the microscope unit 10 and performs predetermined arithmetic processing on the image data generated by the microscope unit 10 to obtain a captured image of the imaging target S. It is a unit to generate.

As schematically shown in FIG. 9, the arithmetic processing unit 20 includes a microscope unit control unit 201, a data acquisition unit 203, a selection unit 205, a captured image reconstruction unit 207, a data output unit 209, A display control unit 211 and a storage unit 213 are mainly provided.

The microscope unit control unit 201 is realized by, for example, a CPU, a ROM, a RAM, a communication device, and the like. The microscope unit control unit 201 generally performs various operations in the microscope unit 10 by transmitting and receiving various control signals to and from the light source, optical element, scanning mechanism, and the like that configure the microscope unit 10 according to the present embodiment. To oversee. Thereby, the light source provided in the microscope unit 10 emits excitation light at a predetermined timing, or the end face of the image guide fiber 103 is scanned in accordance with the scanning method as described above. Further, the light source, the optical element, the scanning mechanism, and the like constituting the microscope unit 10 can also perform various controls in cooperation with each other via the microscope unit control unit 201. Various control information (for example, information on the imaging position) used when the microscope unit control unit 201 controls the microscope unit 10 is output to the data acquisition unit 203, the selection unit 205, and the like as necessary. It is suitably used for various processes in these processing units.

The data acquisition unit 203 is realized by, for example, a CPU, a ROM, a RAM, a communication device, and the like. The data acquisition unit 203 acquires a plurality of image data generated by the microscope unit 10 according to the scanning method as described above from the microscope unit 10. The plurality of image data acquired by the data acquisition unit 203 is transmitted to the selection unit 205 described later. In addition, the data acquisition unit 203 may associate time information related to the date and time when the image data is acquired with the acquired plurality of image data, and store them in the storage unit 213 described later as history information.

The selection unit 205 is realized by, for example, a CPU, a ROM, a RAM, and the like. The selection unit 205 determines, for each of a plurality of pixels constituting the corresponding region of the optical fiber, a pixel value having the maximum luminance among the plurality of image data transmitted from the data acquisition unit 203, as a representative pixel value of the pixel. Select as. Hereinafter, the representative pixel value selection processing performed by the selection unit 205 will be specifically described with reference to FIGS.

Since the image guide fiber 103 is a bundle of a plurality of optical fiber strands 121, as schematically shown in FIG. 10, the interval (arrangement pitch) d between adjacent optical fiber strands or the optical fiber strands Based on the core diameter d ′ of the wire 121, an optical fiber strand corresponding region, which is a region corresponding to one optical fiber strand 121, can be virtually defined. The selection unit 205 according to the present embodiment treats an optical fiber strand corresponding area defined for each optical fiber 121 as a selection target area in a representative pixel value selection process as described in detail below.

The scanning method as shown in FIGS. 8A and 8B is realized in the microscope unit 10, so that, as schematically shown in FIG. 11, the detector provided in the imaging optical system 105 in a certain selection target region. The imaging field of view moves with time. As a result, at a certain moment, in FIG. 1, a situation corresponding to each of the cores C _A to C _E is realized in a certain selection target area.

Here, in the multi-photon excitation process including the two-photon excitation process, as the wave guide mode of light in the optical fiber is lower, it is generated as schematically described with reference to FIG. The fluorescence brightness increases. Further, as the imaging target region of the imaging target at that time is located at the center with respect to the core, the generated fluorescence luminance increases. Therefore, by acquiring images multiple times and selecting the highest fluorescence value, it is possible to selectively acquire information on the fluorescence generated through the multiphoton excitation process by the excitation light that has reached the sample side end surface in the 0th-order mode. Can do. Here, it is considered that the highest fluorescence value is given by image data obtained by imaging a position corresponding to time T = t _MAX in FIG.

Therefore, the selection unit 205 specifies the highest luminance value for each pixel constituting the selection target region with reference to the luminance value at the corresponding position of the plurality of image data including the pixel of interest. In addition, the selection unit 205 uses the specified maximum luminance value as the representative pixel value of the target pixel. For example, as schematically shown in FIG. 12, when N pieces of image data 1 to N exist for a certain pixel of interest, the N pieces of image data are searched across and the maximum luminance value L _MAX is given. The image data is used as the image data of the pixel of interest. In the case of FIG. 12, the image data k is used as image data that gives the representative pixel value of the pixel of interest.

The selection unit 205 performs the above representative pixel value selection process for all the selection target regions (that is, all the optical fiber strand corresponding regions).

Here, since it is considered that the image data giving the maximum brightness value is superimposed with noise accompanying the brightness value, the selection unit 205 has a brightness close to the maximum brightness value instead of the image data giving the maximum brightness value. Image data giving a value may be selected.

In the above description, the case where the selection unit 205 searches across a plurality of generated image data and selects image data giving the highest luminance value has been described. However, the following method is used. However, it is possible to specify a specific value of the maximum luminance value. In other words, the selection unit 205 may identify the maximum luminance value by comparing the data of the peripheral pixels and performing a filtering process that selects the maximum luminance value in the corresponding region of the optical fiber. As an example of such filter processing, for example, an ordinary filter for an area of 10 pixels × 10 pixels can be cited. By using the filter processing, it is possible to search for the highest luminance value in the pixel of interest more quickly and easily.

When the selection unit 205 selects a representative pixel value by the above method, the selection unit 205 outputs information on the selected representative pixel value to the captured image reconstruction unit 207.

The captured image reconstruction unit 207 is realized by, for example, a CPU, a ROM, a RAM, and the like. The captured image reconstruction unit 207 reconstructs a captured image of the imaging target S using the selected representative pixel value. Thereby, it is possible to obtain a captured image of the imaging target S representing the state of fluorescence generated through the multiphoton excitation process.

The captured image reconstruction unit 207 may apply a blur filter typified by a Gaussian filter to the reconstructed captured image of the imaging target S. As a result, it is possible to obtain a captured image in which the selected representative pixel values are connected more smoothly.

Moreover, the captured image reconstruction unit 207 may perform known post-processing other than the above on the reconstructed captured image.

The captured image reconstruction unit 207 outputs the captured image reconstructed as described above to the data output unit 209.

The data output unit 209 is realized by, for example, a CPU, a ROM, a RAM, an output device, a communication device, and the like. The data output unit 209 outputs information on the captured image of the imaging target S output from the captured image reconstruction unit 207 to the display control unit 211. Thereby, information related to the captured image of the imaging target S is output to a display unit (not shown) included in the image acquisition device 1 or an information processing device that can communicate with the image acquisition device 1. Become. Further, the data output unit 209 may output information about the obtained captured image to various recording media and various information processing devices, or output to a paper medium or the like using an output device such as a printer. May be. Further, the data output unit 209 may store the information related to the captured image of the imaging target S in the storage unit 213 or the like as history information in association with time information related to the date and time when the information is calculated.

The display control unit 211 is realized by, for example, a CPU, a ROM, a RAM, an output device, and the like. The display control unit 211 provides various processing results including the captured image of the imaging target S transmitted from the data output unit 209 to an output device such as a display provided in the arithmetic processing unit 20 or outside the arithmetic processing unit 20. Display control when displaying on the output device or the like. Thereby, the user of the image acquisition device 1 can grasp various processing results such as a captured image of the imaging target S on the spot.

The storage unit 213 is realized by, for example, a RAM or a storage device provided in the arithmetic processing unit 20 according to the present embodiment. The storage unit 213 stores various parameters, processing progresses, and various databases and programs that need to be saved when the arithmetic processing unit 20 according to the present embodiment performs some processing. To be recorded. In the storage unit 213, the microscope unit control unit 201, the data acquisition unit 203, the selection unit 205, the captured image reconstruction unit 207, the data output unit 209, the display control unit 211, and the like freely perform data read / write processing. It is possible.

Heretofore, an example of the function of the arithmetic processing unit 20 according to the present embodiment has been shown. Each component described above may be configured using a general-purpose member or circuit, or may be configured by hardware specialized for the function of each component. In addition, the CPU or the like may perform all functions of each component. Therefore, it is possible to appropriately change the configuration to be used according to the technical level at the time of carrying out the present embodiment.

It should be noted that a computer program for realizing each function of the arithmetic processing unit according to the present embodiment as described above can be produced and mounted on a personal computer or the like. In addition, a computer-readable recording medium storing such a computer program can be provided. The recording medium is, for example, a magnetic disk, an optical disk, a magneto-optical disk, a flash memory, or the like. Further, the above computer program may be distributed via a network, for example, without using a recording medium.

<Image acquisition method>
Next, the flow of the image acquisition method performed by the image acquisition device 1 according to this embodiment will be briefly described with reference to FIG. FIG. 13 is a flowchart illustrating an example of the flow of the image acquisition method according to the present embodiment.

In the microscope unit 10 of the image acquisition apparatus 1 according to the present embodiment, excitation light having a predetermined wavelength is applied to the imaging target S by the light source optical system 101 and the image guide fiber 103 under the control of the arithmetic processing unit 20. Irradiation (step S101). As a result, fluorescence due to the multiphoton excitation process is generated in the imaging target S, and an image of the imaging target S due to the fluorescence is transmitted through the image guide fiber 103.

Next, the imaging optical system 105 of the microscope unit 10 scans the end face of the image guide fiber 103 at a predetermined pitch based on the control of the arithmetic processing unit 20 (step S103), and is transmitted to the end face of the image guide fiber 103. A plurality of image data relating to the image of the imaged object S is generated. Thereafter, the imaging optical system 105 outputs the generated plurality of image data to the arithmetic processing unit 20.

The selection unit 205 of the arithmetic processing unit 20 uses a plurality of image data generated by the microscope unit 10 to select a pixel value that gives the maximum luminance as a representative pixel value of the corresponding area of the optical fiber (step S105). Thereafter, the selection unit 205 outputs information regarding the selected representative pixel value to the captured image reconstruction unit 207.

The captured image reconstruction unit 207 reconstructs a captured image of the imaging target S using information on the representative pixel value selected by the selection unit 205 (step S107). In addition, the captured image reconstruction unit 207 performs post-processing such as filter processing using a blur filter on the reconstructed captured image as necessary (step S109).

Thereby, in the image acquisition device 1 according to the present embodiment, a captured image obtained by capturing fluorescence from the imaging target S can be acquired.

The flow of the image acquisition method according to the present embodiment has been briefly described above with reference to FIG.

(About hardware configuration)
Next, the hardware configuration of the arithmetic processing unit 20 according to the embodiment of the present disclosure will be described in detail with reference to FIG. FIG. 14 is a block diagram for describing a hardware configuration of the arithmetic processing unit 20 according to the embodiment of the present disclosure.

The arithmetic processing unit 20 mainly includes a CPU 901, a ROM 903, and a RAM 905. The arithmetic processing unit 20 further includes a host bus 907, a bridge 909, an external bus 911, an interface 913, an input device 915, an output device 917, a storage device 919, a drive 921, and a connection port 923. And a communication device 925.

The CPU 901 functions as an arithmetic processing device and a control device, and controls all or a part of the operation in the arithmetic processing unit 20 according to various programs recorded in the ROM 903, the RAM 905, the storage device 919, or the removable recording medium 927. The ROM 903 stores programs used by the CPU 901, calculation parameters, and the like. The RAM 905 primarily stores programs used by the CPU 901, parameters that change as appropriate during execution of the programs, and the like. These are connected to each other by a host bus 907 constituted by an internal bus such as a CPU bus.

The host bus 907 is connected to an external bus 911 such as a PCI (Peripheral Component Interconnect / Interface) bus via a bridge 909.

The input device 915 is an operation means operated by the user such as a mouse, a keyboard, a touch panel, a button, a switch, and a lever. Further, the input device 915 may be, for example, remote control means (so-called remote control) using infrared rays or other radio waves, or an external connection device such as a mobile phone or a PDA corresponding to the operation of the arithmetic processing unit 20. 929 may be used. Furthermore, the input device 915 includes an input control circuit that generates an input signal based on information input by a user using the above-described operation means and outputs the input signal to the CPU 901, for example. The user of the arithmetic processing unit 20 can input various data and instruct processing operations to the arithmetic processing unit 20 by operating the input device 915.

The output device 917 is a device that can notify the user of the acquired information visually or audibly. Examples of such devices include CRT display devices, liquid crystal display devices, plasma display devices, EL display devices and display devices such as lamps, audio output devices such as speakers and headphones, printer devices, mobile phones, and facsimiles. The output device 917 outputs, for example, results obtained by various processes performed by the arithmetic processing unit 20. Specifically, the display device displays the results obtained by the various processes performed by the arithmetic processing unit 20 as text or images. On the other hand, the audio output device converts an audio signal composed of reproduced audio data, acoustic data, and the like into an analog signal and outputs the analog signal.

The storage device 919 is a data storage device configured as an example of a storage unit of the arithmetic processing unit 20. The storage device 919 includes, for example, a magnetic storage device such as an HDD (Hard Disk Drive), a semiconductor storage device, an optical storage device, or a magneto-optical storage device. The storage device 919 stores programs executed by the CPU 901 and various data, and acoustic signal data and image signal data acquired from the outside.

The drive 921 is a reader / writer for a recording medium, and is built in or externally attached to the arithmetic processing unit 20. The drive 921 reads information recorded on a removable recording medium 927 such as a mounted magnetic disk, optical disk, magneto-optical disk, or semiconductor memory, and outputs the information to the RAM 905. In addition, the drive 921 can write a record on a removable recording medium 927 such as a magnetic disk, an optical disk, a magneto-optical disk, or a semiconductor memory. The removable recording medium 927 is, for example, a DVD medium, an HD-DVD medium, a Blu-ray (registered trademark) medium, or the like. The removable recording medium 927 may be a compact flash (registered trademark) (CompactFlash: CF), a flash memory, or an SD memory card (Secure Digital memory card). Further, the removable recording medium 927 may be, for example, an IC card (Integrated Circuit card) on which a non-contact IC chip is mounted, an electronic device, or the like.

The connection port 923 is a port for directly connecting a device to the arithmetic processing unit 20. Examples of the connection port 923 include a USB (Universal Serial Bus) port, an IEEE 1394 port, a SCSI (Small Computer System Interface) port, and the like. As another example of the connection port 923, there are an RS-232C port, an optical audio terminal, an HDMI (High-Definition Multimedia Interface) port, and the like. By connecting the external connection device 929 to the connection port 923, the arithmetic processing unit 20 acquires the acoustic signal data and the image signal data directly from the external connection device 929, or the acoustic signal data and the image signal to the external connection device 929. Or provide data.

The communication device 925 is a communication interface configured with, for example, a communication device for connecting to the communication network 931. The communication device 925 is, for example, a communication card for a wired or wireless LAN (Local Area Network), Bluetooth (registered trademark), or WUSB (Wireless USB). The communication device 925 may be a router for optical communication, a router for ADSL (Asymmetric Digital Subscriber Line), or a modem for various communication. The communication device 925 can transmit and receive signals and the like according to a predetermined protocol such as TCP / IP, for example, with the Internet or other communication devices. The communication network 931 connected to the communication device 925 is configured by a wired or wireless network, and may be, for example, the Internet, a home LAN, infrared communication, radio wave communication, satellite communication, or the like. .

Heretofore, an example of the hardware configuration capable of realizing the function of the arithmetic processing unit 20 according to the embodiment of the present disclosure has been shown. Each component described above may be configured using a general-purpose member, or may be configured by hardware specialized for the function of each component. Therefore, it is possible to change the hardware configuration to be used as appropriate according to the technical level at the time of carrying out this embodiment.

Subsequently, an image acquisition apparatus and an image acquisition method according to the present disclosure will be specifically described with reference to experimental examples. Note that the following experimental example is merely an example of the image acquisition device and the image acquisition method according to the embodiment of the present disclosure, and the image acquisition device and the image acquisition method according to the present disclosure are limited to the following example. It is not a thing.

In the experimental example shown below, the fluorescence from the fluorescent beads arranged at the tip of the image guide fiber 103 was acquired using the image acquisition device schematically shown in FIG.

FIG. 15 is an explanatory diagram schematically showing the configuration of the microscope unit used in the experimental example. In this experimental example, the light source using the MOPA shown in FIGS. 6C and 6D was used as the light source. The excitation light emitted from the light source is guided to the XY-galvano mirror XY-gal via the lens L and the mirror M, and the imaging position on the image guide fiber 103 is controlled by the galvano mirror. The excitation light that has passed through the galvanometer mirror is guided to the objective lens Obj via the relay lens RL, the mirror M, and the dichroic mirror DM. In this experimental example, a 20 × objective lens having a numerical aperture NA = 0.75 was used as the objective lens Obj.

One end of a commercially available image guide fiber 103 was disposed at the focal position of the objective lens Obj. The image guide fiber 103 is a bundle fiber in which multimode optical fiber strands (core diameter: 3 μm) are arranged at a pitch of 3.5 μm, and the total length is 2 m. Note that the radius of curvature of the bent portion of the image guide fiber 103 was about 15 to 20 cm.

At the other end of the image guide fiber 103, a fluorescent bead group of φ2.1 μm was disposed. The excitation light guided to the fluorescent bead group through the image guide fiber 103 generates fluorescence from the fluorescent bead. The generated fluorescence is guided to a photomultiplier tube as a detector through an image guide fiber 103, an objective lens Obj, a dichroic mirror DM, a lens L, a notch filter F, and the like.

The scanning method shown in FIG. 8A is adopted as the scanning method of the end face of the image guide fiber 103, and the scanning pitch p is 0.1 μm, which is 1/10 or less of the core diameter of the optical fiber. did. The scanning line interval was also set to 0.1 μm. In the obtained image data, one pixel corresponds to 0.1 μm.

When the above fluorescent beads are excited with excitation light having a wavelength of 405 nm, a fluorescence image can be obtained by a one-photon excitation process, and when excited by excitation light having a wavelength of 780 nm, a fluorescence image can be obtained by a two-photon excitation process. . FIG. 16 shows a fluorescence image using excitation light having two wavelengths.

As is clear from FIG. 16, it can be seen that the fluorescence image by the one-photon excitation process is distributed over the entire area of the imaging field where the fluorescent beads are present. On the other hand, in the fluorescence image by the two-photon excitation process, fluorescence images in various states were observed from those having a high fluorescence luminance to portions where no fluorescence was observed. Here, in the two-photon excitation fluorescence image, the one with high fluorescence intensity corresponds to the one excited by the 0th-order mode excitation light, and the excitation light is guided to the center of the fluorescent bead, and the fluorescence intensity is dark It is considered that this corresponds to one excited by higher-order mode excitation light or one in which the excitation light irradiation position is shifted from the center of the fluorescent bead.

A process of selecting a representative pixel value by applying a 10 pixel × 10 pixel two-dimensional ordinary filter to such a two-photon excitation fluorescence image was performed. A fluorescence image reconstructed using the obtained representative pixel values is shown in FIG. 17 together with the original image. As is clear from the reconstructed image shown in FIG. 17, by performing the representative pixel selection process according to this embodiment, it is possible to obtain a fluorescent image by the two-photon excitation process over the entire region where the fluorescent beads are present. did it.

Here, the image guide fiber is excited in a single mode (0th-order mode) at the fiber incident end surface when a bent portion is present in the optical fiber strand or vibration is applied to the optical fiber strand. There is a possibility that the guided light transits to a higher-order mode and the excitation efficiency of the fluorescence due to the multi-photon excitation process decreases. Therefore, in this experimental example, vibration was applied to the image guide fiber during acquisition of image data, and how the obtained image changed was verified.

FIG. 18 shows a one-photon excitation fluorescence image in a case where fluorescence by a one-photon excitation process is imaged ten times in a state where vibration is not applied (hereinafter referred to as “static condition”), and FIG. 1 is shown (hereinafter referred to as “dynamic condition”), a one-photon excitation fluorescence image is shown in a case where fluorescence by a one-photon excitation process is imaged ten times.

As described above, the fluorescence due to the one-photon excitation process has a fluorescence intensity proportional to the intensity of the excitation light. Therefore, no variation in brightness was observed when the captured images were compared under the same conditions. . In addition, even when the captured image under the static condition was compared with the captured image under the dynamic condition, no variation in luminance was observed.

FIG. 20 shows a two-photon excitation fluorescence image obtained by imaging the fluorescence due to the two-photon excitation process ten times under static conditions, and FIG. 21 shows the fluorescence due to the two-photon excitation process ten times under dynamic conditions. A two-photon excitation fluorescence image when imaged is shown.

As is apparent from FIGS. 20 and 21, even in the two-photon excitation fluorescence image, when the respective captured images under the same conditions are compared, variation in luminance is not observed, and the captured image and the motion under the static condition are not observed. Even when the captured images were compared under the target conditions, no variation in luminance was observed.

In FIG. 18 to FIG. 21, five pixels whose fluorescence luminance is not saturated are selected, the maximum value and the minimum value of the fluorescence luminance value in 10 times of imaging are specified, and the value given by the minimum value / maximum value The average value between pixels was calculated. This average value can be considered as an index representing luminance variation. The obtained results are shown in Table 1 below.

As can be seen from Table 1 above, under static conditions, although the variation in luminance of the two-photon excitation fluorescence image is slightly larger than that of the one-photon excitation fluorescence image, it can be understood that the value can withstand practical use. In addition, under dynamic conditions, it can be seen that the variation in luminance is approximately the same between the one-photon excitation fluorescence image and the two-photon excitation fluorescence image.

From the above results, it has been clarified that the fluorescence image obtained by the multiphoton excitation process can be acquired more stably by using the image acquisition method according to the embodiment of the present disclosure. In addition, even when vibration was applied to the image guide fiber, it became clear that single mode (zero-order mode) excitation light generated at the incident side end face can be transmitted in the single mode to the emission side end face. .

The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the technical scope of the present disclosure is not limited to such examples. It is obvious that a person having ordinary knowledge in the technical field of the present disclosure can come up with various changes or modifications within the scope of the technical idea described in the claims. Of course, it is understood that it belongs to the technical scope of the present disclosure.

In addition, the effects described in the present specification are merely illustrative or illustrative, and are not limited. That is, the technology according to the present disclosure can exhibit other effects that are apparent to those skilled in the art from the description of the present specification in addition to or instead of the above effects.

The following configurations also belong to the technical scope of the present disclosure.
(1)
A light source optical system that guides excitation light for generating fluorescence by exciting the imaging target with two or more photons to the imaging target;
A plurality of multimode optical fiber strands are bundled, and the excitation light incident on one end by the light source optical system is transmitted to the imaging target, and the generated in the imaging target An image guide fiber that transmits an image of the imaging target imaged to the other end by fluorescence to the one end;
Scanning the image of the imaged object transmitted to the one end of the image guide fiber at a scanning pitch narrower than the size of the core of the plurality of optical fiber strands, the respective optical fiber strands An imaging optical system that captures at least a part of the corresponding region of the optical fiber corresponding to the above and includes a plurality of images, and generates a plurality of image data of the imaging target,
For each of the plurality of pixels constituting the optical fiber corresponding region, a selection unit that selects a pixel value that has the maximum luminance among the plurality of image data as a representative pixel value of the pixel;
An image acquisition device comprising:
(2)
The image acquisition device according to (1), wherein the scanning pitch is 1/10 or less of a core diameter of the optical fiber.
(3)
The image acquisition device according to (1) or (2), wherein the imaging optical system generates the plurality of image data by imaging the imaging target once while shifting an imaging position for each scanning pitch. .
(4)
The imaging optical system scans the imaging start position in a direction perpendicular to the scanning direction by scanning the imaging position while shifting the imaging position by the arrangement interval of the optical fiber strands and imaging the imaging target. The image acquisition device according to (1) or (2), wherein the plurality of image data are generated by performing the operation a plurality of times while being shifted by a pitch.
(5)
The image acquisition device according to any one of (1) to (4), wherein the selection unit selects the representative pixel value by a filtering process.
(6)
The image acquisition device according to any one of (1) to (5), further including a captured image reconstruction unit that reconstructs a captured image of the imaging target using the selected representative pixel value. .
(7)
The image acquisition device according to (6), wherein the captured image reconstruction unit causes a blur filter to act on the reconstructed captured image.
(8)
The image acquisition device according to any one of (1) to (7), wherein a light source disposed in the light source optical system is a main oscillator having a semiconductor laser and a resonator.
(9)
A light source disposed in the light source optical system is a master oscillator output amplifier (Master Oscillator Power) including a main oscillator having a semiconductor laser and a resonator, and an optical amplifier for amplifying laser light from the main oscillator. The image acquisition device according to any one of (1) to (7), wherein the image acquisition device is Amplifier (MOPA).
(10)
The light source disposed in the light source optical system includes a main oscillator having a semiconductor laser and a resonator, an optical amplifier that amplifies laser light from the main oscillator, and a wavelength that converts the wavelength of the amplified laser light. The image acquisition device according to any one of (1) to (7), wherein the image acquisition device is a light source including a conversion unit.
(11)
Guiding excitation light for generating fluorescence by exciting the imaging target with two or more photons to the imaging target;
The excitation light incident on one end of the image guide fiber is transmitted to the image pickup object by the image guide fiber in which a plurality of multimode optical fiber strands are bundled, and is generated in the image pickup object. Transmitting the image of the imaging target imaged to the other end by the fluorescence to the one end;
Scanning the image of the imaged object transmitted to the one end of the image guide fiber at a scanning pitch narrower than the size of the core of the plurality of optical fiber strands, the respective optical fiber strands Imaging so that at least a part of the corresponding region of the optical fiber corresponding to 1 is included in a plurality of images, and generating a plurality of image data of the imaged object,
For each of the plurality of pixels constituting the corresponding region of the optical fiber, selecting a pixel value having the maximum luminance among the plurality of image data as a representative pixel value of the pixel, and obtaining an image Method.

DESCRIPTION OF SYMBOLS 1 Image acquisition apparatus 10 Microscope unit 20 Arithmetic processing unit 101 Light source optical system 103 Image guide fiber 105 Imaging optical system 121 Optical fiber strand 123 Core 125 Cladding 201 Microscope unit control part 203 Data acquisition part 205 Selection part 207 Captured image reconstruction part 209 Data output unit 211 Display control unit 213 Storage unit

Claims (11)

A light source optical system that guides excitation light for generating fluorescence by exciting the imaging target with two or more photons to the imaging target;
A plurality of multimode optical fiber strands are bundled, and the excitation light incident on one end by the light source optical system is transmitted to the imaging target, and the generated in the imaging target An image guide fiber that transmits an image of the imaging target imaged to the other end by fluorescence to the one end;
Scanning the image of the imaged object transmitted to the one end of the image guide fiber at a scanning pitch narrower than the size of the core of the plurality of optical fiber strands, the respective optical fiber strands An imaging optical system that captures at least a part of the corresponding region of the optical fiber corresponding to the above and includes a plurality of images, and generates a plurality of image data of the imaging target,
For each of the plurality of pixels constituting the optical fiber corresponding region, a selection unit that selects a pixel value that has the maximum luminance among the plurality of image data as a representative pixel value of the pixel;
An image acquisition device comprising: The image acquisition device according to claim 1, wherein the scanning pitch is 1/10 or less of a core diameter of the optical fiber. The image acquisition device according to claim 2, wherein the imaging optical system generates the plurality of image data by imaging the imaging target once while shifting an imaging position for each scanning pitch. The imaging optical system scans the imaging start position in a direction perpendicular to the scanning direction by scanning the imaging position while shifting the imaging position by the arrangement interval of the optical fiber strands and imaging the imaging target. The image acquisition apparatus according to claim 2, wherein the plurality of image data are generated by performing the plurality of times while shifting the pitch. The image acquisition device according to claim 1, wherein the selection unit selects the representative pixel value by a filtering process. The image acquisition apparatus according to claim 1, further comprising a captured image reconstruction unit that reconstructs a captured image of the imaging target using the selected representative pixel value. The image acquisition device according to claim 6, wherein the captured image reconstruction unit causes a blur filter to act on the reconstructed captured image. The image acquisition apparatus according to claim 1, wherein the light source disposed in the light source optical system is a main oscillator having a semiconductor laser and a resonator. The light source disposed in the light source optical system is a master oscillator output amplifier (Master Oscillator Power) composed of a main oscillator having a semiconductor laser and a resonator, and an optical amplifier for amplifying laser light from the main oscillator. The image acquisition device according to claim 1, which is an amplifier (MOPA). The light source disposed in the light source optical system includes a main oscillator having a semiconductor laser and a resonator, an optical amplifier that amplifies laser light from the main oscillator, and a wavelength that converts the wavelength of the amplified laser light. The image acquisition device according to claim 1, wherein the image acquisition device is a light source including a conversion unit. Guiding excitation light for generating fluorescence by exciting the imaging target with two or more photons to the imaging target;
The excitation light incident on one end of the image guide fiber is transmitted to the image pickup object by the image guide fiber in which a plurality of multimode optical fiber strands are bundled, and is generated in the image pickup object. Transmitting the image of the imaging target imaged to the other end by the fluorescence to the one end;
Scanning the image of the imaged object transmitted to the one end of the image guide fiber at a scanning pitch narrower than the size of the core of the plurality of optical fiber strands, the respective optical fiber strands Imaging so that at least a part of the corresponding region of the optical fiber corresponding to 1 is included in a plurality of images, and generating a plurality of image data of the imaged object,
For each of a plurality of pixels constituting the region corresponding to the optical fiber, selecting a pixel value having the maximum luminance among the plurality of image data as a representative pixel value of the pixel;
Including an image acquisition method.

Images