JP2018197881A - Induced radiation suppression microscope device - Google Patents

Abstract

To provide an STED microscope device that can increase the convenience of a user in terms of a resolution and a required time.SOLUTION: An STED microscope device 1A includes: an STED light source 11 generating an STED light LS; an excitation light source 12 generating an excitation light LE; an optical system 15A for emitting a phase-modulated SLM13 presenting a phase pattern for forming an STED light LSinto a ring shape by phase modulation, and for emitting an excitation light LEand a phase modulated STED light LSto an observation target region; a detector 16 for detecting fluorescence PL generated from the observation target region; and a controller 17a for controlling the phase pattern. Changing the phase pattern by the controller 17a enables the inner diameter of the ring of the STED light LSafter phase modulation to be changed.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a stimulated radiation suppression microscope apparatus.

  Non-Patent Document 1 describes a technique related to a stimulated emission suppression (STED) microscope. In the STED microscope described in this document, an annular STED beam is generated using a phase modulation type spatial light modulator.

JP2013-92687A

Travis J. Gould et al., “Adaptive optics enables 3D STED microscopy in aberrating specimens”, OPTICS EXPRESS, Vol.20, No. 19, pp.20998-21009, 2012

  Currently, so-called super-resolution microscopes have been developed that acquire images with a resolution below the optical diffraction limit. Various techniques have been proposed as a super-resolution technique used in the super-resolution microscope, and one of them is a STED microscope. The STED microscope irradiates an observation object almost simultaneously with laser light as observation excitation light (hereinafter referred to as an excitation beam) and short-pulse laser light for stimulated emission (hereinafter referred to as a STED beam). It is possible to locally generate fluorescence from the observation object.

  The principle of the STED microscope is shown below. FIG. 18A and FIG. 18B are diagrams showing the principle of fluorescence generation. As shown in FIG. 18A, when the observation object is irradiated with excitation light LE having an excitation wavelength, electrons are excited from the ground state to the excited state (arrow A1 in the figure). Thereafter, electrons transit from the excited state to the ground state within a few microseconds (arrow A2 in the figure), and at this time, fluorescence PL having a wavelength corresponding to the energy difference between the ground state and the excited state is generated. .

  On the other hand, the STED light LS shown in FIG. 18B is irradiated to the observation object with a predetermined time difference after the above-described excitation light LE is irradiated. The electrons that have been excited by the excitation light LE are induced by the STED light LS and transition to the ground state (arrow A3 in the figure). At this time, since the energy corresponding to the wavelength of the STED light LS transits, the wavelength of the generated light LA becomes equal to the wavelength of the STED light LS. By such an action, in the region irradiated with the STED light LS after being irradiated with the excitation light LE, light LA having a wavelength different from that of the fluorescent PL is generated instead of the fluorescent PL. Further, by setting the predetermined time difference to the nano order, a time difference can be given between the generation timing of the fluorescence PL and the generation timing of the light LA.

  FIG. 19 is a diagram illustrating an example of the shapes of (a) excitation light LE, (b) STED light LS, and (c) fluorescence PL. In the STED microscope, as shown in FIG. 19A, first, a circular excitation light LE is irradiated onto a certain observation target region. Thereafter, as shown in FIG. 19B, an annular STED light LS is irradiated on the circular region. As a result, the generation of the fluorescence PL is suppressed in the annular region, and as shown in FIG. 19 (c), the region near the center of the annular shape (region surrounded by the annular shape) Fluorescence PL can be generated only from a small area, and an image can be acquired with a resolution below the diffraction limit.

In the above-described STED microscope, the resolution is determined by the diameter _{D 2} of fluorescent PL shown in FIG. 19 (c). To improve the resolution, but may be smaller in diameter D _2, Then becomes longer the time required for the entire observation area to scan (scan). Further, when larger the diameter D ₂ in order to reduce the time required to scan the entire observation area (scan), the resolution is lowered. For this reason, in the conventional STED microscope, there is a problem that only one of the improvement of the resolution and the reduction of the required time can be realized, and the convenience is lacking.

  The present invention has been made in view of such problems, and an object of the present invention is to provide a STED microscope apparatus that can improve user convenience with respect to resolution and time required.

  In order to solve the above-described problem, a STED microscope apparatus according to the present invention includes a STED light source that generates STED light, an excitation light source that generates excitation light, and a first STED light that is shaped into an annular shape by phase modulation. A phase modulation type first spatial light modulator in which a phase pattern of the above is presented, an optical system for irradiating the observation target region with excitation light and STED light after phase modulation, and fluorescence generated from the observation target region A detector for detecting and a control unit for controlling the first phase pattern, and the control unit changes the first phase pattern, whereby the inner diameter of the ring of the STED light after phase modulation can be changed. It is characterized by being.

  In the above STED microscope apparatus, the STED light output from the STED light source is formed into an annular shape by phase modulation in the first spatial light modulator. The annular STED light is irradiated to the observation target region after the excitation light is irradiated to the observation target region. Thereby, the generation of fluorescence is suppressed in the annular region, and the fluorescence is generated only from the region surrounded by the annular STED light. Therefore, according to the STED microscope apparatus, it is possible to acquire an image with a resolution below the diffraction limit.

  In the above STED microscope apparatus, the inner diameter of the ring of the STED light after phase modulation can be changed by changing the phase pattern by the control unit. Accordingly, the inner diameter of the ring can be reduced when the resolution needs to be improved, and the inner diameter of the ring can be increased when it is desired to shorten the time required for scanning the entire observation target region. As described above, according to the STED microscope apparatus, the convenience of the user can be improved with respect to the resolution and the required time.

  The STED microscope apparatus further includes a second spatial light modulator that presents a second phase pattern for shaping the excitation light into a circular shape by phase modulation, and the control unit includes the second phase light modulator. The pattern may be further controlled. Thereby, the shape of excitation light can be arbitrarily controlled.

  In the STED microscope apparatus, the control unit divides the STED light and irradiates the plurality of regions with the pattern superimposed on the first phase pattern, and divides the excitation light and irradiates the plurality of regions. It is good also as the pattern for making it overlap on a 2nd phase pattern. Thereby, it is possible to observe a plurality of regions at the same time, and the required time can be further shortened.

  In the above STED microscope apparatus, the detector may be a two-dimensional detector. In this case, the STED microscope apparatus further includes an optical scanner that scans the fluorescence on the light receiving surface of the two-dimensional detector, and the irradiation time intervals of the excitation light and the stimulated emission suppression light are determined by the pixels on the light receiving surface in the scanning direction. Depending on the width and the scanning speed of the optical scanner, each pixel may be set not to receive fluorescence multiple times. Thereby, the resolution can be improved.

  In the STED microscope apparatus, the first phase pattern is a pattern in which the phase increases from 0 (rad) to 2π × n (rad) (n is an integer equal to or greater than 1) in a spiral shape around a certain point. , And the control unit may change the inner diameter of the annular ring by changing the integer n. For example, the inner diameter of the ring of the STED light after phase modulation can be suitably changed by such a first phase pattern.

  In the STED microscope apparatus described above, the first phase pattern spirally increases in phase from 0 (rad) to 2π (rad) around a certain point (n is an integer of 1 or more). It may be characterized in that the inner diameter of the annulus can be changed by changing the integer n, including a repeating pattern. For example, the inner diameter of the ring of the STED light after phase modulation can be suitably changed by such a first phase pattern.

  In the STED microscope apparatus, the control unit has a storage unit that stores a plurality of patterns respectively corresponding to a plurality of inner diameters of the ring of the induced radiation suppression light, and the pattern selected from the plurality of patterns May be included in the first phase pattern. As a result, the user can easily change the first phase pattern according to the desired resolution and required time.

  The STED microscope apparatus further includes an input unit for inputting a desired value of the inner diameter of the annular ring, and the first phase pattern spirals around a certain point from 0 (rad) to m (rad). It may include a pattern in which the phase increases to (m is a real number of 2π or more), and the control unit sets the real number m based on a desired value of the inner diameter of the circular ring input from the input unit. For example, the inner diameter of the ring of the STED light after phase modulation can be suitably changed by such a first phase pattern.

  According to the STED microscope apparatus of the present invention, it is possible to improve the convenience of the user with respect to the resolution and the required time.

It is a block diagram which shows the structure of the STED microscope apparatus which concerns on 1st Embodiment of this invention. (A) It is a figure which shows the cross-sectional shape perpendicular | vertical to the optical axis of the STED light input into SLM. (B) It is a figure which shows the cross-sectional shape perpendicular | vertical to the optical axis of STED light output from SLM. (A)-(d) It is a figure which shows notionally the pattern for shape | molding STED light in the ring shape contained in the phase pattern for STED light shaping | molding shown by SLM13. (A)-(d) It is the figure which represented the phase value in each pixel of the pattern shown by FIG. 3 with the shading of the color. (A)-(d) It is a figure which shows the shape of the STED light obtained by each pattern shown by FIG. 3 being shown by SLM. It is a flowchart which shows operation | movement of a STED microscope apparatus. It is a flowchart which shows the process of such a control part. It is a flowchart which shows the process of a control part. It is a block diagram which shows the structure of the STED microscope apparatus as a comparative example. It is a figure which shows notionally the pattern for shape | molding STED light in a ring shape contained in the phase pattern for STED light shaping | molding shown by SLM. (A), (b) It is a figure which shows notionally the pattern for shape | molding STED light in a ring shape contained in the phase pattern for STED light shaping | molding shown by SLM. (C) It is a figure which shows the pattern as a comparative example. (A)-(c) It is a figure which shows typically the annular | circular shape of STED light in the case of m = 2 (pi), 2 (pi) <m <4 (pi), and m = 4 (pi). (A) It is a figure which shows the some excitation light produced | generated by dividing excitation light. (B) It is a figure which shows the several STED light produced | generated by dividing STED light. (C) It is a figure which shows several fluorescence. It is a figure which shows an example of the pattern for dividing | segmenting STED light and producing | generating several STED light. It is a block diagram which shows the structure of a STED microscope apparatus as a 4th modification. (A) It is a figure which shows typically a mode that the fluorescence image formation point is scanned in the light-receiving surface of the two-dimensional imaging device of a 4th modification. (B) It is a graph which shows the irradiation timing of excitation light and STED light. It is a block diagram which shows the structure of a STED microscope apparatus as a 5th modification. (A), (b) It is a figure which shows the generation principle of fluorescence. It is a figure which shows an example of each shape of (a) excitation light, (b) STED light, and (c) fluorescence.

  Hereinafter, embodiments of a STED microscope apparatus according to the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

  FIG. 1 is a block diagram showing a configuration of a STED microscope apparatus 1A according to the first embodiment of the present invention. The STED microscope apparatus 1A is an apparatus for obtaining a fluorescence image of the observation object B such as a cell specimen. As shown in FIG. 1, the STED microscope apparatus 1A of the present embodiment includes a STED light source 11, an excitation light source 12, spatial light modulators (SLM) 13 and 14, an optical system 15A, a detector 16, and An arithmetic control device 17 is provided.

The STED light source 11 is a light source that generates the STED light LS ₁ . The STED light source 11 is a pulse light source that outputs light having high coherency such as laser light. Examples of the STED light source 11 include a lamp light source, a laser light source such as a laser diode, or an LED. The wavelength of the STED light LS ₁ is an arbitrary wavelength that does not overlap the peak wavelength of the fluorescent PL generated in the observation object B and is included in the wavelength band of the fluorescent PL. Note that the STED light source 11 may not be a pulse light source, and may be configured by, for example, a combination of a light source that outputs continuous light (CW light) and an optical shutter or pulse modulation AOM (Acousto-Optic Modulator).

The excitation light source 12 is a light source that generates excitation light LE ₁ . The excitation light source 12 is a pulse light source that outputs light having high coherence such as laser light. Examples of the excitation light source 12 include a laser light source such as a laser diode, or an LED. The wavelength of the excitation light LE ₁ is a wavelength that includes the excitation wavelength of the fluorescent substance contained in the observation object B described later. The excitation light source 12 may not be a pulsed light source, and may be configured by a combination of a light source that outputs continuous light (CW light) and an optical shutter or pulse modulation AOM, for example.

The SLM 13 is a phase modulation type SLM, which modulates the phase of the input light at each part of the two-dimensional modulation surface and outputs the light after phase modulation. The SLM 13 is a first spatial light modulator in the present embodiment. The SLM 13 receives the STED light LS ₁ from the STED light source 11 and outputs the modulated STED light LS ₂ . The SLM 13 controls the condensing irradiation conditions such as the condensing position, condensing intensity, and condensing shape of the STED light LS ₂ by presenting the phase pattern (kinoform) obtained by numerical calculation on the modulation surface.

FIG. 2A is a diagram showing a cross-sectional shape perpendicular to the optical axis of the STED light LS ₁ input to the SLM 13. FIG. 2B is a diagram showing a cross-sectional shape perpendicular to the optical axis of the STED light LS ₂ output from the SLM 13. In this embodiment, a circular STED light LS ₁ as shown in FIG. 2A is input to the SLM 13, and the SLM 13 converts the STED light LS ₂ into an inner diameter as shown in FIG. 2B by phase modulation. A STED light shaping phase pattern (first phase pattern) for forming an annular shape having D ₁ is presented. This STED light shaping phase pattern is controlled by a pattern signal SP ₁ sent from the control unit 17 a of the arithmetic control device 17. The SLM 13 may be either a reflection type or a transmission type. As the SLM 13, for example, a refractive index changing material type SLM is preferably used. Examples of the refractive index changing material type SLM include an LCOS (Liquid Crystal on Silicon) type SLM, an LCD (Liquid Crystal Display), an electric address type liquid crystal element, an optical address type liquid crystal element, and a variable mirror type SLM (Segment Mirror type SLM, Continuous Deformable Mirror SLM).

Refer to FIG. 1 again. The SLM 14 is a second spatial light modulator in the present embodiment. The SLM 14 receives the excitation light LE ₁ from the excitation light source 12, and outputs the modulated excitation light LE ₂ . The SLM 14 controls the condensing irradiation conditions such as the condensing position, condensing intensity, and condensing shape of the excitation light LE ₂ by presenting the phase pattern (kinoform) obtained by numerical calculation on the modulation surface. In the present embodiment, the SLM 14 presents an excitation light shaping phase pattern (second phase pattern) for shaping the excitation light LE ₂ into a circular shape (see FIG. 19A) by phase modulation. The excitation light molding phase pattern is controlled by the pattern signal SP ₂ sent from the control unit 17a. The SLM 14 may be a phase modulation type or an intensity modulation (amplitude modulation) type. Examples of the phase modulation type SLM include those similar to the SLM 13 described above. The SLM 14 may be either a reflection type or a transmission type. Further, a DOE (Diffractive Optical Element) may be provided instead of the SLM 14. In the following description, the case where the SLM 14 is a phase modulation type will be mainly described.

The optical system 15A is provided to irradiate the observation target region of the observation target B with the excitation light LE ₂ and the STED light LS ₂ . The optical system 15A includes dichroic mirrors 15a and 15b, an optical scanner 15c, an objective lens 15d, and an imaging optical system 15e.

The dichroic mirror 15a reflects light in a wavelength band including the wavelength of the STED light LS _2, transmits light in the wavelength band including the wavelength of fluorescence PL that occurs in observation object B. The dichroic mirror 15a is disposed on the optical axis connecting the objective lens 15d and the imaging optical system 15e. The dichroic mirror 15a receives the STED light LS ₂ from SLM 13, for reflecting the STED light LS ₂ in the observation object B. Further, the dichroic mirror 15a transmits the fluorescence PL from the observation object B.

The dichroic mirror 15b reflects light in a wavelength band including the wavelength of the excitation light LE ₂ and transmits STED light LS ₂ and light in a wavelength band including each wavelength of the fluorescence PL generated in the observation object B. The dichroic mirror 15b is disposed on the optical axis connecting the objective lens 15d and the dichroic mirror 15a. The dichroic mirror 15b receives the excitation light LE ₂ from SLM 14, is reflected toward the observation object B the pumping light LE _2. The dichroic mirror 15b passes through the fluorescence PL from STED light LS ₂ and the observation object B from the dichroic mirror 15a.

Optical scanner 15c is on the observation object B, and a device for scanning the focusing position of the STED light LS ₂ and the excitation light LE _2. The optical scanner 15c is disposed on the optical axis connecting the objective lens 15d and the dichroic mirror 15b. As the optical scanner 15c, for example, a galvano, a resonant, a polygon mirror, a MEMS (Micro Electro Mechanical System) mirror, or an acousto-optic element (AOM or AOD (Acousto-Optic Deflector)) is preferably used.

Objective lens 15d is located above the observation object B, is irradiated on the observation target B while condensing the STED light LS ₂ and the excitation light LE _2. At this time, these optical axes are controlled by the SLMs 13 and 14 so that the optical axes of the STED light LS ₂ and the excitation light LE ₂ coincide with each other. The objective lens 15d collimates the fluorescence PL generated in the observation object B by the irradiation of the excitation light LE _2.

  The imaging optical system 15e is disposed on the optical axis between the dichroic mirror 15a and the detector 16. The imaging optical system 15e receives the fluorescence PL collimated by the objective lens 15d and passed through the dichroic mirrors 15a and 15b, and forms an image of the fluorescence PL on the detection surface of the detector 16.

The detector 16 detects the light intensity of the fluorescence PL imaged by the imaging optical system 15e. As the detector 16, for example, a one-dimensional detection such as an optical sensor for detecting light intensity at one point such as a photomultiplier tube, a photodiode or an avalanche photodiode, an area image sensor such as a CCD image sensor or a CMOS image sensor, or a line sensor. A multi-anode photomultiplier tube is preferably used. The detector 16 provides a light intensity signal SD ₁ indicating the light intensity of the fluorescence PL to the arithmetic and control unit 17. In particular, when a multi-anode photomultiplier tube or an area image sensor is used, if a multipoint is generated by the SLM 13 and the SLM 14 as in a fourth modification described later, the fixed multipoint that is less affected by scanning is used. The fluorescence PL can be detected by the detector 16, and the scanning time can be reduced. In the case of using a multi-anode photomultiplier tube, it is preferable that the gain can be adjusted for each detection unit.

The arithmetic and control unit 17 is configured by a computer having a CPU and a memory, for example. The arithmetic control device 17 includes a control unit 17a and an image processing unit 17b. The control unit 17 a controls the first phase pattern presented to the SLM 13 and the second phase pattern presented to the SLM 14. That is, the control unit 17a, based on the desired inner and outer diameters of the STED light LS ₁ of the wavelength or annular shape of the STED light LS _2, determines the STED light molding phase pattern presented to the SLM 13. In addition, the control unit 17a determines the excitation light shaping phase pattern presented to the SLM 14 based on the wavelength of the excitation light LE ₁ and the circular desired diameter of the excitation light LE ₂ . Since the STED light LS ₁ and the excitation light LE ₁ have different wavelengths, the phase pattern is designed according to each wavelength. More specifically, the STED light shaping phase pattern presented on the SLM 13 is designed based on the wavelength of the STED light LS ₁ , and the excitation light shaping phase pattern presented on the SLM 14 is designed based on the excitation light LE _1. Has been.

More specifically, the control unit 17a is formed with a phase pattern phi _Kinoform kinoform designed to provide a phase distribution for condensing STED light LS ₂ to a predetermined position, a STED light LS ₂ in an annular shape A sum φ _SLM = φ _kinoform + φ _pat with the condensing control pattern φ _pat to cause the _SLM 13 to present the phase pattern for STED light shaping. Similarly, the control unit 17a, the kinoform designed to provide a phase distribution for condensing the excitation light LE ₂ at a predetermined position and the phase pattern phi _Kinoform, for molded excitation light LE ₂ in a circular shape A sum φ _SLM = φ _kinoform + φ _pat with the light collection control pattern φ _pat is presented to the _{SLM 14} as an excitation light shaping phase pattern. If the phase of the light input to the phase type SLM is φ _in and the phase value given to the phase type SLM is φ _SLM , the phase φ _{out of the} modulated light to be output is φ _out = φ _SLM + φ _in Become.

Further, in the STED microscope, it is necessary to irradiate the observation object B with the STED light LS ₂ with a predetermined time difference after irradiating the excitation light LE ₂ . Therefore, the control section 17a, after the excitation light source 12 is irradiated with pulsed excitation light LE _1, after the predetermined time has elapsed, as STED light source 11 illuminates the STED light LS ₁ pulsed The light emission timings of the STED light source 11 and the excitation light source 12 are controlled. Further, the control unit 17a controls the optical scanner 15c to scan the condensing positions of the excitation light LE ₂ and the STED light LS ₂ on the observation object B.

The image processing unit 17b inputs the light intensity signal SD ₁ from the detector 16. The image processing unit 17b creates a fluorescence image based on the light intensity of the fluorescence PL detected by the detector 16 and the light collection position by the optical scanner 15c. The fluorescence image created by the image processing unit 17b is displayed on the display device 19.

In the present embodiment, by changing the control section 17a of the STED light molding phase pattern presented to the SLM 13, STED light LS ₂ of the circular ring inside diameter _{D 1} (see FIG. 2 (b)) It can be changed. This point will be described below.

3A to 3D conceptually show patterns P _{11 to} P ₁₄ for forming the STED light LS ₂ into an annular shape, which are included in the STED light shaping phase pattern presented on the SLM ₁₃ . FIG. 4 (a) to 4 (d) represent the phase values in the pixels of the patterns P _{11 to} P ₁₄ shown in FIGS. 3 (a) to 3 (d), respectively, by color shading. In the figure, the lighter the color, the closer the phase value is to 0 (rad), and the darker the color, the closer the phase value is to 2π (rad). As shown in FIGS. 3 and 4, these patterns P _{11 to} P ₁₄ spirally increase the phase from 0 (rad) to 2π (rad) n times (n Is an integer of 1 or more). 3A and 4A show the case where n = 1, FIGS. 3B and 4B show the case where n = 2, and FIGS. 3C and 4C show the case where n = 2. FIG. 3D and FIG. 4D show the case where n = 3, and the case where n = 4, respectively. The patterns P _{11 to} P ₁₄ are so-called Laguerre Gaussian (LG) beam phase patterns. Such patterns P _{11 to} P ₁₄ can also be expressed using Laguerre polynomials. Note that the gradation width from 0 (rad) to 2π (rad) in the STED light shaping phase pattern is set according to the wavelength of the STED light LS ₁ .

Each of FIGS. 5A to 5D is a diagram illustrating the shape of the STED light LS ₂ obtained by presenting each of the patterns P _{11 to} P ₁₄ described above to the SLM 13. 5 (a) to 5 (d), the light intensity is indicated by the shade of color. The light intensity is lighter and the light intensity is lighter and the light intensity is darker. Referring to FIGS. 5A to 5D, it can be seen that the inner diameter of the ring of the STED light LS ₂ increases as the repetition number n increases. That is, by changing the control unit 17a repeatedly n, it is possible to change the inner diameter D ₁ of the circle ring shown in FIG. 2 (b).

Control unit 17a, for example, may have a storage unit 17c for storing in advance a plurality of patterns of repetition number corresponding to a plurality of the inner diameter D ₁ of the annular (see Figure 1). In that case, the control unit 17a includes an input device 18 according to the desired inner diameter D ₁ inputted from the (input unit, see FIG. 1) to select a pattern corresponding to the inner diameter D _1, STED light shaping phase Superimpose on the pattern. Alternatively, the control unit 17a according to the desired inner diameter D ₁ inputted from the input device 18, calculates the number of repetitions n feasible the inner diameter D _1, a STED light forming a pattern based on the repetition number n You may superimpose on a phase pattern.

The operation of the STED microscope apparatus 1A of the present embodiment described above will be described. FIG. 6 is a flowchart showing the operation of the STED microscope apparatus 1A. As shown in the figure, first, information regarding the STED light LS ₁ and the excitation light LE ₁ is input to the arithmetic and control unit 17 via the input device 18 (step S1). Next, the control unit 17a of the arithmetic control device 17 determines the STED light shaping phase pattern and the excitation light shaping phase pattern (step S2).

Then, by an instruction from the control unit 17a, the excitation light source 12 emits excitation light LE ₁ (step S31). The STED light source 11 emits the STED light LS _{1 in accordance} with an instruction from the control unit 17a after a predetermined time from the emission of the excitation light LE ₁ (step S41). The excitation light LE ₁ is shaped into a circular shape by the SLM 14 (step S32), and the STED light LS ₁ is shaped into an annular shape by the SLM 13 (step S42). The shaped excitation light LE ₂ is irradiated to the observation target region of the observation target B through the dichroic mirror 15b, the optical scanner 15c, and the objective lens 15d (step S33). Thereafter, after the predetermined time, the shaped STED light LS ₂ is irradiated on the observation target region of the observation target B through the dichroic mirror 15a, the optical scanner 15c, and the objective lens 15d (step S43). Thereby, generation | occurrence | production of fluorescence PL is suppressed in an annular | circular shaped area | region, and fluorescence PL generate | occur | produces only from the area | region enclosed by this annular | circular shape (refer FIG.19 (c)). Subsequently, the light intensity of the fluorescence PL is detected by the detector 16 (step S5). A light intensity signal SD ₁ relating to the light intensity of the fluorescence PL is sent from the detector 16 to the arithmetic and control unit 17.

After step S5 is completed, the condensing position of the STED light LS ₂ and the excitation light LE ₂ on the observation object B is moved by the optical scanner 15c. And said step S2-S5 is performed again. As described above, the intensity of the fluorescence PL in a wide region of the observation object B is detected by alternately repeating the movement of the light collecting position and the above steps S2 to S5. Subsequently, a fluorescence image is created in the image processing unit 17b of the arithmetic and control unit 17 (step S6). This fluorescent image is displayed by the display device 19 (step S7).

  In order to acquire a three-dimensional fluorescent image, it is preferable to change the distance between the objective lens 15d and the observation object B after steps S1 to S6 and perform steps S1 to S6 again. By repeating such an operation a plurality of times, a planar tomographic image is accumulated. Thereafter, the image processing unit 17b may perform a 3D image reconstruction process based on the accumulated data. In addition, the depth was continuously changed by repeating the operation | movement which measured the image acquisition of multiple times using a counter etc., and moved the observation target B to an optical axis direction, and changed observation depth in multiple times. A three-dimensional image may be reconstructed by acquiring an image group and interpolating (linear interpolation, spline, etc.) between the images.

The control unit 17a may store a plurality of patterns _P 11 _{to P 14} corresponding to a plurality of the inner diameter _{D 1} of the annular pre in response to an input from the input device 18, these patterns _{P 11} ~ You may select the appropriate pattern from the P _14. FIG. 7 is a flowchart showing the processing of the control unit 17a. As shown in FIG. 7, first, a desired operation mode is selected by the user from a plurality of operation modes (scan modes) corresponding to a plurality of scanning times or a plurality of resolutions (step S11). Next, the control unit 17a, from the pattern _P 11 _{to P 14,} selects a pattern corresponding to the selected operation mode (step S12). Subsequently, the control unit 17a creates a STED light shaping phase pattern including the selected pattern, and causes the SLM 13 to present the STED light shaping phase pattern (step S13).

Alternatively, the control unit 17a may create the STED light shaping phase pattern by the following process. FIG. 8 is a flowchart showing the processing of the control unit 17a. As shown in FIG. 8, first, the desired inner diameter D ₁ is input by the user (step S21). Next, the control unit 17a according to the desired inner diameter _{D 1,} and calculates the number of repetitions n feasible the inside diameter _{D 1} (step S22). Then, the control unit 17a creates a pattern based on the calculated repetition number n (step S23), creates a STED light shaping phase pattern including the pattern, and causes the SLM 13 to present the STED light shaping phase pattern ( Step S24).

The effects obtained by the STED microscope apparatus 1A of the present embodiment described above will be described. In the STED microscope apparatus 1 </ b> A, the STED light LS ₁ output from the STED light source 11 is formed into an annular shape by phase modulation in the SLM 13. The ring-shaped STED light LS ₂ is irradiated with the excitation light LE _{2 on the} observation target region, and then overlapped on the observation target region. Thus, in the annular region generating fluorescent PL is suppressed, fluorescent PL is generated only from the region surrounded by the STED light LS ₂ annular. Therefore, according to the STED microscope apparatus 1A of the present embodiment, it is possible to acquire an image with a resolution below the diffraction limit.

FIG. 9 is a block diagram showing a configuration of a STED microscope apparatus 100 as a comparative example. The STED microscope apparatus 100 is not provided with a SLM13,14, and a phase plate 102 for forming the STED light LS ₂ annularly. In such a configuration, in order to change the inner diameter D ₁ of the annular shape of the STED light LS _2, it is necessary to replace the phase plate 102 to another phase plate. In contrast, in the STED microscope apparatus 1A of the present embodiment, a control unit 17a changes the phase pattern, the inner diameter _{D 1} of the annular of STED light LS ₂ after phase modulation is changeable. Thus, when the improved resolution is required to reduce the inner diameter D ₁ of the circle ring, to increase the inner diameter D ₁ of the annular when you want to shorten the time needed for the entire observation area to scan (scan) Can do. As described above, according to the STED microscope apparatus 1A of the present embodiment, it is possible to improve the convenience for the user regarding the resolution and the required time.

Further, as in the present embodiment, the STED microscope apparatus 1A includes the SLM 14 for shaping the excitation light LE ₂ into a circular shape by phase modulation, and the control unit 17a presents the excitation light shaping phase pattern presented on the SLM 14. May be controlled. Thereby, the shape of the excitation light LE ₂ can be arbitrarily and easily controlled.

As shown in FIGS. 3 and 4, the STED optical shaping phase pattern repeats the increase in phase from 0 (rad) to 2π (rad) in a spiral manner around a certain point A n times. includes any pattern P ₁₁ to P _14, the control unit 17a is by varying the integer n, it may be possible to change the inner diameter D ₁ of the annular. For example, the inner diameter D ₁ of the ring of the STED light LS ₂ after phase modulation can be suitably changed by such a STED light shaping phase pattern.

Also, as in the present embodiment, the control unit 17a includes a storage unit 17c for storing a plurality of patterns respectively corresponding to the STED light circle multiple inner diameter _{D 1} of the ring of the LS _2, the selected pattern STED It may be included in the photoforming phase pattern. Thereby, the user can easily change the phase pattern for STED light shaping according to the desired resolution and required time.

  Further, as in the present embodiment, the detector 16 may detect light via the optical scanner 15c. Thereby, a confocal image can be acquired without using a confocal pinhole as in a general confocal microscope apparatus.

(First modification)
FIG. 10 is a diagram conceptually showing a pattern P ₁₅ for shaping the STED light LS ₂ into an annular shape, which is included in the STED light shaping phase pattern presented on the SLM 13. In the above embodiment, the control unit 17a, instead of the pattern _P 11 _{to P 14} illustrated in FIG. 3, the pattern _{P 15} shown in FIG. 10, is superimposed on STED light molding phase pattern presented to SLM13 May be.

As shown in FIG. 10, this pattern P ₁₅ is a pattern in which the phase increases from 0 (rad) to 2π × n (rad) (n is an integer of 1 or more) in a spiral manner around a certain point A. is there. For example, in the case of n = 2, the pattern P ₁₅ is a pattern in which the phase increases from 0 (rad) to 4π (rad) spirally around the point A. the inner diameter _{D 1} of the is equal to the inner diameter _{D 1} of the annular implemented by helically 0 (rad) is repeated 2 times the phase increase of up to 2 [pi (rad) from the pattern (see Figure 3 (b)). The pattern P ₁₅ is a so-called Laguerre Gaussian (LG) beam phase pattern. Such patterns P ₁₅ can be expressed even with a Laguerre polynomial.

As in the present modification, the STED optical shaping phase pattern includes a pattern P ₁₅ whose phase increases from 0 (rad) to 2π × n (rad) in a spiral manner around a certain point A, and includes a control unit 17a. There by changing the integer n, may be possible to change the inner diameter D ₁ of the annular. For example, the inner diameter D ₁ of the ring of the STED light LS ₂ after phase modulation can be suitably changed by such a STED light shaping phase pattern. In addition, when the phase modulation width of the SLM 13 is 0 to 2π (rad), as in the patterns P _{11 to} P ₁₄ illustrated in FIG. 3, spirally from 0 (rad) to 2π (rad). It is preferable to use a pattern in which the phase increase is repeated n times (that is, the pattern is folded at the phase 2π).

(Second modification)
FIG. 11A is a diagram conceptually showing a pattern P ₁₆ for forming the STED light LS ₂ into an annular shape, which is included in the STED light shaping phase pattern presented on the SLM 13. In the above embodiment, the control unit 17a, instead of the pattern _P 11 _{to P 14} illustrated in FIG. 3, the pattern _{P 16} shown in FIG. 11 (a), STED light molding phase is presented in SLM13 pattern You may superimpose on.

As shown in FIG. 11A, this pattern P ₁₆ is a pattern in which the phase increases from 0 (rad) to m (rad) (m is a real number of 2π or more) spirally around a certain point A. It is. For example, when m = 3π, the pattern P ₁₆ is a pattern in which the phase increases from 0 (rad) to 3π (rad) spirally around the point A. STED light molding phase pattern includes such a pattern P _16, by the control unit 17a changes the real m, it can be suitably changed inside diameter D ₁ of the annular. In this modified example, the control unit 17a according to the desired inner diameter D ₁ inputted from the input device 18, calculates a real number m that can realize the internal diameter D _1, a STED light forming a pattern based on the real number m Superimpose on the phase pattern.

Note that, as shown in FIG. 11B, the pattern of this modification example has a spiral shape whose phase increases from 0 (rad) to 2π (rad), then turns back at 2π (rad), and then returns to 0 ( The pattern P ₁₇ may increase the phase from rad) to π (rad). The inner diameter D ₁ of the annulus realized by the pattern P ₁₇ is equal to the inner diameter D _{1 of the} annulus realized by the pattern P ₁₆ whose phase increases from 0 (rad) to 3π (rad) spirally. . However, as shown in FIG. 11C, after the phase increases spirally from 0 (rad) to 1.5π (rad), it is turned back at π (rad) and again from 0 (rad) to 1. In the pattern P ₁₈ in which the phase increases to 5π (rad), the STED light LS ₂ cannot be formed into an annular shape.

FIG. 12A to FIG. 12C are diagrams schematically showing the annular shape of the STED light LS ₂ when m = 2π, 2π <m <4π, and m = 4π, respectively. As shown in FIG. 12, as the smaller the inner diameter D ₁ of the annular value of m is small, the inner diameter D ₁ of the annular as the value of m is large is increased. Therefore, the control unit 17a changes the real m, can be suitably changed inside diameter D ₁ of the annular.

(Third Modification)
In the above-described embodiment, the control unit 17a divides the excitation light LE ₁ and further superimposes a pattern for irradiating the divided excitation light LE ₂ on the plurality of regions simultaneously with the excitation light shaping phase pattern. Also good. FIG. 13A is a diagram showing a plurality of excitation lights LE ₂ generated by dividing the excitation light LE ₁ . In this case, the control unit 17a divides the STED light LS ₁ into a plurality of patterns, and a pattern for irradiating the divided STED light LS ₂ to a plurality of regions of the observation object B simultaneously as a STED light shaping phase pattern. Furthermore, it is good to superimpose.

FIG. 13B is a diagram showing a plurality of STED lights LS ₂ generated by dividing the STED light LS ₁ . If STED light LS ₂ shown in FIG. 13 (b) is irradiated immediately after the excitation light LE ₂ shown in FIG. 13 (a) is irradiated, as shown in FIG. 13 (c), a plurality of observation object B Fluorescence PL is simultaneously generated from the region. In FIG. 13A to FIG. 13C, the light intensity is shown in shades of color. The light intensity is lighter and the light intensity is lighter and the light intensity is darker.

FIG. 14 is a diagram illustrating an example of a pattern for dividing the STED light LS ₁ to generate a plurality of STED light LS ₂ . In FIG. 14, the phase value in each pixel is represented by color shading. The lighter the color, the closer the phase value is to 0 (rad), and the darker the color, the closer the phase value is to 2π (rad). In this modification, for example, a pattern as shown in FIG. 14 is superimposed on a pattern for forming an annular shape (for example, patterns P _{11 to} P ₁₄ shown in FIG. 3), thereby forming an annular shape. multiple STED light LS ₂ is generated. The pattern for dividing the excitation light LE ₁ to generate a plurality of excitation lights LE ₂ is also superimposed on the pattern for shaping the excitation light LE ₂ into a circular shape. Note that the pattern shown in FIG. 14 is designed based on the wavelength of the STED light LS ₁ .

  According to this modification, it is possible to simultaneously observe a plurality of regions of the observation object B. Therefore, since the scanning time by the optical scanner 15c can be reduced, the time required for observation can be further shortened.

(Fourth modification)
FIG. 15 is a block diagram showing a configuration of a STED microscope apparatus 1B as a fourth modification of the embodiment. The difference between the STED microscope apparatus 1B of this modified example and the STED microscope apparatus 1A of the above-described embodiment is the imaging method of the fluorescence PL. That is, the STED microscope apparatus 1B of the present modification includes an optical system 15B and a two-dimensional imaging device 20 instead of the optical system 15A and the detector 16 of the above embodiment. In addition, the other structure in STED microscope apparatus 1B is the same as that of the said embodiment.

The optical system 15B is provided to irradiate the observation target region of the observation target B with the excitation light LE ₂ and the STED light LS ₂ . The optical system 15B includes dichroic mirrors 15a and 15b, an optical scanner 15c, an objective lens 15d, a dichroic mirror 15f, and an imaging optical system 15g. The configurations of the dichroic mirrors 15a and 15b, the optical scanner 15c, and the objective lens 15d are the same as those in the above embodiment.

The dichroic mirror 15f reflects the light of the wavelength band including the wavelength of fluorescence PL that occurs in observation object B, and transmits light in a wavelength band including the wavelength of the STED light LS ₂ wavelengths and the excitation light LE _2. In this modification, the wavelength of the STED light LS ₂ is set to a wavelength between the wavelength of the fluorescence PL and the wavelength of the excitation light LE ₂ . The dichroic mirror 15f is disposed on the optical axis connecting the objective lens 15d and the optical scanner 15c. The dichroic mirror 15 f receives the fluorescence PL from the observation object B and reflects the fluorescence PL toward the two-dimensional imaging device 20. The imaging optical system 15g is disposed between the dichroic mirror 15f and the two-dimensional imaging device 20, receives the fluorescence PL reflected by the dichroic mirror 15f, and binds the fluorescence PL on the detection surface of the two-dimensional imaging device 20. Image.

The two-dimensional imaging device 20 detects the light intensity of the fluorescence PL imaged by the imaging optical system 15g. As the two-dimensional imaging device 20, for example, an area image sensor such as a CCD image sensor or a CMOS image sensor is preferably used. Two-dimensional imaging device 20, an optical image signal SD ₂ showing the optical image of the fluorescent PL, is provided to the arithmetic and control unit 17. The image processing unit 17b of the arithmetic control device 17 creates a fluorescence image based on the light image of the fluorescence PL captured by the two-dimensional imaging device 20. The fluorescence image created by the image processing unit 17b is displayed on the display device 19.

FIG. 16A is a diagram schematically illustrating a state where the imaging point of the fluorescence PL is scanned on the light receiving surface 20a of the two-dimensional imaging device 20 of the present modification. In FIG. 16A, the scanning direction of the fluorescence PL is represented by a solid arrow. The light-receiving surface 20a has a plurality of pixels 20b arranged in a two-dimensional form with M rows and N columns (M and N are integers of 2 or more). The optical scanner 15c controls the irradiation positions of the excitation light LE ₂ and the STED light LS ₂ so that the fluorescence PL is scanned in the pixel 20b in the row direction, and the next row is scanned after the completion of the scanning of one row. . Note that the exposure time of the two-dimensional imaging device 20 is set to the time from the start of scanning of the fluorescence PL to the end of scanning.

FIG. 16B is a graph showing the irradiation timings of the excitation light LE ₂ and the STED light LS ₂ , and the horizontal axis corresponds to the scanning time of one line shown in FIG. In this modification, in order to improve the resolution, it is desirable that one pixel 20b avoids receiving the fluorescence PL a plurality of times. Accordingly, the pulse time interval T between the excitation light LE ₂ and the STED light LS ₂ depends on the width of the pixel 20b in the scanning direction, the scanning speed of the optical scanner 15c, and the imaging magnification of the objective lens 15d and the imaging optical system 15g. It is desirable to set.

Specifically, as shown in FIG. 16B, first, when the optical axis of the fluorescence PL is positioned at the first pixel 20b of the row, the excitation light LE ₂ and the STED light LS ₂ are continuously generated. The fluorescence PL generated at that time is incident on the first pixel 20b. Subsequently, after the optical axes of the excitation light LE ₂ and the STED light LS ₂ are moved by the optical scanner 15c so that the optical axis of the fluorescence PL is positioned at the next pixel 20b in the row, the excitation light LE ₂ and STED light LS ₂ is continuously irradiated with, then the fluorescent PL generated enters the next pixel 20b. By repeating such an operation over a plurality of pixels 20b in the row, a fluorescence image for one row is obtained. Then, by repeating such an operation over a plurality of lines, a single fluorescent image is obtained.

(Fifth modification)
FIG. 17 is a block diagram showing a configuration of a STED microscope apparatus 1C as a fifth modification of the embodiment. The difference between the STED microscope apparatus 1C of this modified example and the STED microscope apparatus 1A of the above embodiment is that the SLM 14 that receives the excitation light LE ₁ from the excitation light source 12 and outputs the modulated excitation light LE ₂ is not used. is there. That is, the STED microscope apparatus 1C of the present modification includes the mirror 22 and the dichroic mirror 23 so that the optical axis of the excitation light LE ₁ from the excitation light source 12 and the optical axis of the STED light LS ₁ from the STED light source 11 are obtained. The SLM 13 receives both the STED light LS ₁ and the excitation light LE ₁ . The other configurations in the STED microscope apparatus 1C are the same as those in the above embodiment.

Excitation light LE ₁ from the excitation light source 12 is reflected by the mirror 22 and input to the dichroic mirror 23. The dichroic mirror 23 is transmitted through the wavelength of the STED light LS _1, since the reflected excitation light LE _1, and the optical axis of the STED light LS ₁ from the optical axis and the STED light source 11 of the excitation light LE ₁ from the excitation light source 12 The SLM 13 receives the excitation light LE ₁ and the STED light LS ₁ . Here, when the SLM 13 is a spatial light modulator that modulates only light of a specific polarization, such as an LCOS-SLM, for example, the polarization of the STED light LS _{1 is} the specific polarization, and the polarization of the excitation light LE ₁ is By making the polarized light orthogonal to the specific polarized light, only the STED light LS ₁ can be modulated. More specifically, when the SLM 13 is an LCOS-SLM, the LCOS-SLM can only perform phase modulation on a polarization component (for example, a horizontal polarization component) in the same direction as the alignment direction of the liquid crystal. Therefore, when the STED light LS ₁ to be phase-modulated so as to have an annular shape is horizontally polarized light and the excitation light LE ₁ that does not require phase modulation is vertically polarized light, these are coaxially combined and input to the SLM 13 in advance. , STED light LS ₁ is phase-modulated and output as STED light LS ₂ . On the other hand, the excitation light LE ₁ is not modulated and is output as the excitation light LE ₁ . According to the configuration of the modified STED microscope apparatus 1C, it is possible to guide light between the STED light source 11 and the excitation light source 12 and the SLM 13 using, for example, a polarization fiber that preserves polarized light. The light source 11 and the excitation light source 12 and the optical system 15A can be physically separated. Thereby, the influence on the optical system 15A due to vibration and heat generated in the STED light source 11 and the excitation light source 12 can be reduced, the optical system 15A can be downsized and stabilized, and the STED light source 11 and the excitation light source 12 can be easily changed. become.

DESCRIPTION OF SYMBOLS 1A ... STED microscope apparatus, 1B ... STED microscope apparatus, 11 ... STED light source, 12 ... Excitation light source, 13, 14 ... SLM, 15A, 15B ... Optical system, 15a, 15b ... Dichroic mirror, 15c ... Optical scanner, 15d ... Objective Lens, 15e ... Imaging optical system, 15f ... Dichroic mirror, 15g ... Imaging optical system, 16 ... Detector, 17 ... Calculation control device, 17a ... Control unit, 17b ... Image processing unit, 17c ... Storage unit, 18 ... input device, 19 ... display, 20 ... two-dimensional imaging device, 20a ... light-receiving surface, 20b ... pixels, B ... observation object, _LE 1, LE 2 _... pumping _light, LS _1, LS 2 ... STED light, PL ... fluorescence.

In order to solve the above-described problem, a STED microscope apparatus according to the present invention includes a STED light source that generates STED light, an excitation light source that generates excitation light, and a first STED light that is shaped into an annular shape by phase modulation. A phase modulation type first spatial light modulator in which a phase pattern of the first spatial light modulator is presented, and an optical scanner for controlling a condensing position in the observation target region of the stimulated emission suppression light phase-modulated by the first spatial light modulator; an optical system for irradiating the STED light after the excitation light and the phase modulation on the observation target region, a detector for detecting fluorescence generated from the observation target region, and a control unit for controlling the first phase pattern, the In addition, the inner diameter of the ring of the STED light after the phase modulation can be changed by the control unit changing the first phase pattern.

In the above STED microscope apparatus, the detector may be a two-dimensional detector. In that case, the optical scanner may be characterized Rukoto by scanning the fluorescence on the light receiving surface of the two-dimensional detector. Further, in that case, the irradiation time intervals of the excitation light and the induced radiation suppression light are set so that each pixel does not receive the fluorescence multiple times according to the width of the pixel on the light receiving surface in the scanning direction and the scanning speed of the optical scanner. It may be characterized by being. Thereby, the resolution can be improved.

The STED microscope apparatus may be characterized in that the detector detects fluorescence via an optical scanner. In the STED microscope apparatus, the first phase pattern is a pattern in which the phase increases from 0 (rad) to 2π × n (rad) (n is an integer equal to or greater than 1) in a spiral shape around a certain point. , And the control unit may change the inner diameter of the annular ring by changing the integer n. For example, the inner diameter of the ring of the STED light after phase modulation can be suitably changed by such a first phase pattern.

Claims (9)

A stimulated emission suppression light source that generates stimulated emission suppression light; and
An excitation light source that generates excitation light;
A first spatial light modulator of a phase modulation type in which a first phase pattern for shaping the induced radiation suppression light into an annular shape by phase modulation is presented;
An optical system for irradiating the observation target region with the excitation light and the stimulated emission suppression light after phase modulation;
A detector for detecting fluorescence generated from the observation target region;
A control unit for controlling the first phase pattern;
With
An induced radiation suppression microscope apparatus, wherein an inner diameter of a ring of the induced radiation suppression light after phase modulation can be changed by the control unit changing the first phase pattern. A second spatial light modulator on which a second phase pattern for shaping the excitation light into a circular shape by phase modulation is presented;
The stimulated radiation suppression microscope apparatus according to claim 1, wherein the control unit further controls the second phase pattern.   The controller is configured to superimpose a pattern for dividing the stimulated emission suppression light and irradiating a plurality of regions on the first phase pattern, and to divide the excitation light and irradiate the plurality of regions. The induced radiation suppression microscope apparatus according to claim 2, wherein the pattern is superimposed on the second phase pattern.   The stimulated radiation suppression microscope apparatus according to claim 3, wherein the detector is a two-dimensional detector. An optical scanner that scans the fluorescence on the light receiving surface of the two-dimensional detector;
The irradiation time interval of the excitation light and the stimulated emission suppression light is set so that each pixel does not receive the fluorescence a plurality of times according to the width of the pixel on the light receiving surface in the scanning direction and the scanning speed of the optical scanner. The stimulated radiation suppression microscope apparatus according to claim 4, wherein: The first phase pattern includes a pattern in which a phase increases from 0 (rad) to 2π × n (rad) (n is an integer of 1 or more) spirally around a certain point,
The induction radiation suppression microscope apparatus according to any one of claims 1 to 5, wherein an inner diameter of the annular ring can be changed by the control unit changing an integer n. The first phase pattern includes a pattern in which a phase increase from 0 (rad) to 2π (rad) is repeated n times (n is an integer of 1 or more) spirally around a certain point,
The induction radiation suppression microscope apparatus according to any one of claims 1 to 5, wherein an inner diameter of the annular ring can be changed by the control unit changing an integer n.   The control unit includes a storage unit that stores a plurality of patterns respectively corresponding to a plurality of inner diameters of the annular ring of the induced radiation suppression light, and a pattern selected from the plurality of patterns is stored in the first phase. The induced radiation suppression microscope apparatus according to claim 1, wherein the microscope is included in a pattern. An input unit for inputting a desired value of the inner diameter of the ring,
The first phase pattern includes a pattern in which the phase increases from 0 (rad) to m (rad) (m is a real number greater than or equal to 2π) spirally around a certain point;
6. The stimulated emission suppression according to claim 1, wherein the control unit sets a real number m based on a desired value of the inner diameter of the annular ring input from the input unit. Microscope device.