JP2013142854A - Non-linear microscope and non-linear observation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a non-linear microscope with an evaluation function of performance, and a non-linear observation method.SOLUTION: A non-linear microscope illustrating the present invention includes calculation means (29) for calculating a coherent transmission function of the non-linear microscope in the non-linear microscope including: an illumination optical system (19) which collects illumination light to be supplied from a light source (11) on an object (10) for observation and generates a coherent non-linear optical process at its light collection spot; and a detection optical system (20) which detects coherent signal light generated in the non-linear optical process at the light collection spot.

Description

  The present invention relates to a nonlinear microscope and a nonlinear observation method.

  The confocal microscope has an irradiation optical system for condensing illumination light emitted from a light source toward a test object, and a confocal microscope in which light emitted from the test object is arranged at a position conjugate with the light collection point. And a detection optical system that detects through a focus stop (pinhole member) (see, for example, Patent Document 1).

  In this configuration, since most of the light emitted from the layer with a height different from the condensing point of the illumination light is blocked by the pinhole member, the illumination optical system is focused on the layer with the desired height, and the illumination is performed. If detection is repeated while two-dimensionally scanning the object with a light spot, an image (cross-sectional image) of the layer can be acquired.

  Furthermore, it is possible to obtain a three-dimensional image of the test object by repeating the acquisition of the cross-sectional image while changing the height of the focal point with respect to the test object, and synthesizing the cross-sectional images acquired at each height position . In addition, what is detected as signal light in a normal confocal microscope is fluorescence emitted from the test object with an intensity linear with respect to the intensity of the irradiation light.

  On the other hand, in recent years, development of nonlinear microscopes using a coherent nonlinear optical process has been actively performed (see, for example, Patent Document 2). What is detected as signal light in the nonlinear microscope is light emitted from the test object with a non-linear intensity with respect to the intensity of the illumination light.

  Since this nonlinear microscope can use light having a relatively long wavelength (near infrared light) as illumination light, it is possible to observe the deep portion of the test object. Further, since the nonlinear optical process occurs only in a minute region near the focal point of the objective lens, the resolution of the nonlinear microscope is much higher than the resolution of the linear microscope.

JP 2007-279085 A JP 2009-47435 A

  However, although the performance of the nonlinear microscope is empirically known, there are few examples that quantitatively represent it.

  Accordingly, an object of the present invention is to provide a nonlinear microscope and a nonlinear observation method having a performance evaluation function.

  A non-linear microscope exemplifying the present invention condenses illumination light supplied from a light source on an object to be observed, and an illumination optical system that causes a coherent non-linear optical process at the condensing point; A non-linear microscope comprising a detection optical system for detecting coherent signal light generated in the non-linear optical process, comprising a calculating means for calculating a coherent transfer function of the non-linear microscope.

  A non-linear observation method exemplifying the present invention includes an illumination procedure for condensing illumination light supplied from a light source on an object to be observed and causing a coherent non-linear optical process at the condensing point; A non-linear observation method including a detection procedure for detecting coherent signal light generated in the non-linear optical process, including a calculation procedure for calculating a coherent transfer function of the non-linear observation method.

  According to the present invention, a nonlinear microscope and a nonlinear observation method having a performance evaluation function are realized.

It is a block diagram of the nonlinear microscope of this embodiment. Each of a plurality of different types of nonlinear optical processes is represented by a double-sided Feynman diagram. It is a figure explaining the signal detection method in SRL and SRG. It is a figure explaining a typical three-dimensional pupil function. It is a figure which shows binarization CTF for every type of nonlinear optical process.

[First Embodiment]
Hereinafter, a nonlinear microscope will be described as a first embodiment of the present invention.

  FIG. 1 is a configuration diagram of the nonlinear microscope of the present embodiment. The upper part of FIG. 1 is a diagram showing a configuration on the light source side of the nonlinear microscope, and the lower part of FIG. 1 is a diagram showing a configuration on the sample side of the nonlinear microscope.

  As shown in the upper part of FIG. 1, the nonlinear microscope of the present embodiment includes a laser light source 11, a lens 121, a beam splitter 122, optical parametric oscillators (OPO) 125-1 and 125-2, and an electro-optic modulator ( EOM) 126-1 and 126-2, total reflection mirrors 123, 127A, 127B, and 127C, an optical path length adjustment mechanism 128, and a beam splitter 129 are provided.

  As shown in the lower part of FIG. 1, the nonlinear microscope of this embodiment includes a dichroic mirror 14, an optical scanner 15, a relay optical system (lenses 16 </ b> A and 16 </ b> B), an objective lens switching mechanism 19, and a transmission stage 100. , Objective lens switching mechanism 20, total reflection mirror 21, filter switching mechanisms 22t and 22r, condenser lenses 25t and 25r, pinhole masks 26t and 26r, photodetectors 27t and 27r, and lock-in amplifier 23t. , 23r, a control unit 28, and a calculation unit 29 are provided.

  The stage 100 is a transmission type stage and supports the culture vessel 10 which is a transparent vessel. A transparent culture solution containing living cells is accommodated inside the culture vessel 10. Molecules (proteins, lipids, etc.) contained in the living cells become observation objects of the nonlinear microscope.

  The laser light source 11 is a pulse laser light source that oscillates pulse laser light, and the pulse shape of the pulse laser light oscillated by the laser light source is set to an appropriate shape. With this setting, the energy density of the central portion (high-density spot) of the light spot described later becomes an energy density suitable for causing a nonlinear optical process to occur in the observation target. The pulsed laser light emitted from the laser light source 11 enters the lens 121.

  The pulse laser beam that has entered the lens 121 becomes a parallel light beam having a large diameter, and is branched into a pulse laser beam L that enters the beam splitter 122 and passes through the beam splitter 122, and a pulse laser beam L that reflects from the beam splitter 122. (In FIG. 1, the same reference numerals are assigned to light having the same optical frequency.)

  The pulsed laser light L that has passed through the beam splitter 122 enters the beam splitter 129 through the OPO 125-1, the EOM 126-1, the total reflection mirror 127A, the optical path length difference adjusting mechanism 128, and the total reflection mirror 127B in this order. Further, the pulsed laser light L reflected from the beam splitter 122 enters the beam splitter 129 through the total reflection mirror 123, the OPO 125-2, the EOM 126-2, and the total reflection mirror 127C in this order.

  Hereinafter, the pulse laser beam L passing through the OPO 125-1 is referred to as “pulse laser beam L1”, and the pulse laser beam L passing through the OPO 125-2 is referred to as “pulse laser beam L2”.

OPO125-1 placed into a single optical path of the pulsed laser light L1, by converting the optical frequency of the pulsed laser light L1 incident to set the optical frequency of the pulsed laser light L1 to a predetermined frequency omega _1. Note that the output frequency ω _{1 of the} OPO 125-1 is appropriately switched by the control unit 28.

OPO125-2 placed into a single optical path of the pulsed laser beam L2 by converting the optical frequency of the pulsed laser beam L2 incident, set to a different frequency omega ₂ is an optical frequency of the pulsed laser light L2 and omega ₁ . The output frequency ω _{2 of the} OPO 125-2 is appropriately switched by the control unit 28 (hereinafter referred to as ω ₁ > ω ₂ ).

  The EOM 126-1 disposed on the single optical path of the pulse laser beam L1 modulates the intensity of the incident pulse laser beam L1 in a time direction at a predetermined frequency. The modulation frequency of the EOM 126-1 is set to a value lower than the repetition frequency of the pulse laser beam L1. Note that the modulation function of the EOM 126-1 is turned on / off at an appropriate timing by the control unit 28, and the pulse laser beam L1 is in an unmodulated state in a state where the modulation function of the EOM 126-1 is turned off. Further, if the output intensity of the EOM 126-1 is set to zero, the pulse laser beam L1 can be turned off.

  The EOM 126-2 disposed in the single optical path of the pulse laser beam L2 modulates the intensity of the incident pulse laser beam L2 at a predetermined frequency in the time direction. The modulation frequency of the EOM 126-2 is set to a value lower than the repetition frequency of the pulse laser beam L2. Note that the modulation function of the EOM 126-2 is turned on / off at an appropriate timing by the control unit 28, and the pulse laser beam L2 is in an unmodulated state in a state where the modulation function of the EOM 126-2 is turned off. Further, if the output intensity of the EOM 126-2 is set to zero, the pulse laser beam L1 can be turned off.

  The optical path length difference adjusting mechanism 128 is a mechanism for adjusting the optical path length difference between the pulse laser beams L1 and L2. The optical path length difference between them is determined in advance so that the timing at which any pulse of the pulse laser beam L1 enters the beam splitter 129 matches the timing at which any pulse of the pulse laser beam L2 enters the beam splitter 129. It has been adjusted.

  The pulse laser beams L1 and L2 incident on the beam splitter 129 are integrated with each other and travel toward the dichroic mirror 14. These pulse laser beams L1 and L2 pass through the dichroic mirror 14 and enter the effective objective lens of the objective lens switching mechanism 19 through the optical scanner 15 and the lenses 16A and 16B in this order.

  The objective lens switching mechanism 19 includes a plurality of objective lenses having different NAs, and selectively sets one of the objective lenses to the optical path. Here, the objective lens set in the optical path by the objective lens switching mechanism 19 is referred to as an “effective objective lens”. The switching position of the objective lens switching mechanism 19 is controlled by the control unit 28.

  The pulsed laser beams L1 and L2 that have entered the effective objective lens of the objective lens switching mechanism 19 are condensed to form a light spot on the observation target surface 10A of the culture vessel 10. In the central portion (high density spot) of the light spot, a nonlinear optical process occurs, and signal light is generated.

  Of the signal light generated in the culture vessel 10, the light emitted to the effective objective lens side of the objective lens switching mechanism 20 (transmitted signal light) enters the effective objective lens of the objective lens switching mechanism 20.

  The objective lens switching mechanism 20 includes a plurality of objective lenses having different NAs, and selectively sets one of the objective lenses to the optical path. Here, the objective lens set in the optical path by the objective lens switching mechanism 20 is referred to as an “effective objective lens”. The switching position of the objective lens switching mechanism 20 is controlled by the control unit 28.

  The signal light incident on the effective objective lens of the objective lens switching mechanism 20 enters the effective filter of the filter switching mechanism 22t via the total reflection mirror 21.

  The filter switching mechanism 22t includes a plurality of filters having different passbands, and selectively sets one of these filters to the optical path. Here, the filter set by the filter switching mechanism 22t in the optical path is referred to as an “effective filter”. The switching position of the filter switching mechanism 22t is controlled by the control unit 28.

  The transmitted signal light Lr that has passed through the effective filter of the filter switching mechanism 22t is collected by the condenser lens 25t and travels to the vicinity of the pinhole of the pinhole mask 26t. The transmitted signal light Lt that has passed through the pinhole mask 26t enters the photodetector 27t and is converted into an electrical signal. This electrical signal is input to the control unit 28 via the lock-in amplifier 23t.

  The lock-in amplifier 23t is synchronously controlled with the EOM 125-1 or EOM 125-2 by the control unit 28, and changes at the same frequency as the modulation frequency of the EOM 125-1 or EOM 125-2 among the electrical signals output from the photodetector 27t. Extract the components to be used. Note that the lock-in function of the lock-in amplifier 23t is turned on / off by the control unit 28 at an appropriate timing.

  Of the signal light generated in the culture vessel 10, the light emitted to the effective objective lens side of the objective lens switching mechanism 19 (reflected signal light) enters the effective objective lens of the objective lens switching mechanism 19.

  The reflected signal light incident on the effective objective lens of the objective lens switching mechanism 19 enters the dichroic mirror 14 through the lenses 16B and 16A and the optical scanner 15 in this order. This reflected signal light enters the effective filter of the filter switching mechanism 22r.

  The filter switching mechanism 22r includes a plurality of filters having different passbands, and selectively sets one of these filters to the optical path. Here, the filter set by the filter switching mechanism 22r in the optical path is referred to as an “effective filter”. The switching position of the filter switching mechanism 22r is controlled by the control unit 28.

  The reflected signal light Lr that has passed through the effective filter of the filter switching mechanism 22r is collected by the condenser lens 25r and travels to the vicinity of the pinhole of the pinhole mask 26r. The reflected signal light Lr that has passed through the pinhole mask 26r enters the photodetector 27r and is converted into an electrical signal. This electric signal is input to the control unit 28 via the lock-in amplifier 23r.

  The lock-in amplifier 23r is synchronously controlled with the EOM 125-1 or EOM 125-2 by the control unit 28, and changes at the same frequency as the modulation frequency of the EOM 125-1 or EOM 125-2 among the electrical signals output from the photodetector 27r. Extract the components to be used. Note that the lock-in function of the lock-in amplifier 23r is turned on / off by the control unit 28 at an appropriate timing.

  The optical scanner 15 is an optical scanner having a pair of galvanometer mirrors and the like. When the optical scanner 15 is driven, the above-described light spot moves on the observation target surface 10A.

  The control unit 28 drives the optical scanner 15 to perform two-dimensional scanning on the observation target surface 10A with the light spot, and drives the light detector 27r when the light spot is at each scanning position, thereby reflecting the reflected signal light. Distribution (reflection observation image) on the observation target surface 10A with a high intensity can be acquired. Furthermore, the control unit 28 acquires a three-dimensional reflection observation image by repeatedly acquiring the reflection observation image while shifting the culture vessel 10 in the optical axis direction. This reflection observation image is displayed on a monitor (not shown) via the calculation unit 29.

  Prior to the display of the reflection observation image, the arithmetic unit 29 performs a process for calculating the resolution related to the reflection observation image (resolution calculation process) and a process for sharpening the reflection observation image (sharpening process) (for details). Later).

  Further, the control unit 28 moves the stage 100 in the direction (xy direction) perpendicular to the optical axis to perform two-dimensional scanning on the observation target surface 10A with the light spot, and when the light spot is at each scanning position. By driving the photodetector 27t, the distribution (transmission observation image) of the intensity of the transmitted signal light on the observation target surface 10A can be acquired. Furthermore, the control unit 28 acquires a three-dimensional transmission observation image by repeatedly acquiring the transmission observation image while shifting the culture vessel 10 in the optical axis direction. The transmission observation image is displayed on a monitor (not shown) via the calculation unit 29.

  Prior to the display of the transmission observation image, the calculation unit 29 performs a process for calculating the resolution regarding the transmission observation image (resolution calculation process) and a process for sharpening the transmission observation image (sharpening process) (details will be described later). ).

  The control unit 28 switches the photodetector to be driven between the photodetectors 27r and 27t in accordance with an instruction from the user, and switches the mechanism to be driven between the optical scanner 15 and the stage 100. By switching at, the observation image acquired by the nonlinear microscope is switched between the transmission observation image and the reflection observation image. Thus, switching the observation image acquired by the nonlinear microscope between the transmission observation image and the reflection observation image corresponds to switching the illumination type of the nonlinear microscope between the transmission type and the reflection type.

Further, the control unit 28 switches the wavelength used in the nonlinear optical process by switching the combination of the optical frequency ω ₁ of the pulsed laser light L ₁ and the optical frequency ω ₂ of the pulsed laser light L ₂ in accordance with an instruction from the user. .

  Further, in accordance with an instruction from the user, the control unit 28 turns on / off the pulse laser beams L1 and L2, on / off states of the modulation functions of the EOMs 126-1 and 126-2, the lock-in amplifier 23t, By switching the combination of the ON / OFF state of the lock-in function of 23r and the setting positions of the filter switching mechanisms 22t and 22r, the type of the nonlinear optical process used for observation is switched. The types of non-linear optical processes that can be switched are, for example, the following types (A) to (H).

(A) First harmonic (SHG): a nonlinear optical process in which illumination light with an optical frequency ω ₁ is irradiated and signal light with an optical frequency ωs = 2ω ₁ is detected. This SHG is represented by a double-sided Feynman diagram in FIG. Therefore, in order to use this SHG, the pulse laser beam L1 having the optical frequency ω ₁ is turned on, the pulse laser beam L2 having the optical frequency ω ₂ is turned off, and both the EOMs 126-1 and 126-2 are used. off the modulation function, the lock-in amplifier 23t, turns off both the lock-in feature of 23r, may be set filter passband is .omega.s = 2 [omega ₁ filter switching mechanism 22t, the effective filter 22r.

(B) Sum frequency (SFG): a nonlinear optical process in which illumination light with frequencies ω ₁ and ω ₂ is irradiated and signal light with a frequency ωs = (ω ₁ + ω ₂ ) is detected. FIG. 2B shows this SFG by a double-sided Feynman diagram. Therefore, in order to use this SFG, both the pulse laser beam L1 having the optical frequency ω ₁ and the pulse laser beam L2 having the optical frequency ω ₂ are turned on, and the EOMs 126-1 and 126-2 are turned on. Both modulation functions are turned off, both lock-in amplifiers 23t and 23r are turned off, and a filter whose passband is ωs = (ω ₁ + ω ₂ ) is set as an effective filter of the filter switching mechanisms 22t and 22r. do it.

(C) Difference frequency (DFG): a nonlinear optical process in which illumination light with frequencies ω ₁ and ω ₂ is irradiated and signal light with a frequency ωs = (ω ₁ −ω ₂ ) is detected (where ω ₁ > ω ₂ ). . This DFG is represented by a double-sided Feynman diagram in FIG. Therefore, in order to use this DFG, both the pulse laser beam L1 having the optical frequency ω ₁ and the pulse laser beam L2 having the optical frequency ω ₂ are turned on, and the EOMs 126-1 and 126-2 are turned on. Both modulation functions are turned off, both lock-in functions of the lock-in amplifiers 23t and 23r are turned off, and a filter whose passband is ωs = (ω ₁ −ω ₂ ) is used as an effective filter of the filter switching mechanisms 22t and 22r. You only have to set it.

(D) Second high frequency (THG): a nonlinear optical process in which illumination light with a frequency ω ₁ is irradiated and signal light with a frequency ωs = 3ω ₁ is detected. This THG is represented by a double-sided Feynman diagram in FIG. Therefore, in order to use this THG, the pulse laser beam L1 having the optical frequency ω ₁ is turned on, the pulse laser beam L2 having the optical frequency ω ₂ is turned off, and both the EOMs 126-1 and 126-2 are used. off the modulation function, the lock-in amplifier 23t, turns off both the lock-in feature of 23r, may be set filter passband is .omega.s = 3 [omega] ₁ filter switching mechanism 22t, the effective filter 22r.

(E) Coherent anti-Stokes Raman scattering (CARS): a nonlinear optical process (ω ₁ > ω ₂ ) in which illumination light with frequencies ω ₁ and ω ₂ is irradiated and signal light with a frequency ωs = (2ω ₁ −ω ₂ ) is detected. ). This CARS is represented by a double-sided Feynman diagram in FIG. Therefore, in order to use this CARS it includes a pulsed laser light L1 optical frequency is omega _1, and on both the pulsed laser beam L2 optical frequency is omega _2, of EOM126-1,126-2 Both modulation functions are turned off, both lock-in functions of the lock-in amplifiers 23t and 23r are turned off, and a filter whose passband is ωs = (2ω ₁ −ω ₂ ) is used as an effective filter of the filter switching mechanisms 22t and 22r. You only have to set it.

(F) Coherent Stokes Raman scattering (CSRS): a nonlinear optical process in which illumination light with frequencies ω ₁ and ω ₂ is irradiated and signal light with a frequency ωs = (2ω ₂ −ω ₁ ) is detected (where ω ₁ > ω ₂ , 2ω ₂ > ω ₁ ). This CSRS is represented by a double-sided Feynman diagram in FIG. Therefore, in order to use this CSRS, both the pulse laser beam L1 having the optical frequency ω ₁ and the pulse laser beam L2 having the optical frequency ω ₂ are turned on, and the EOMs 126-1 and 126-2 are turned on. Both modulation functions are turned off, both lock-in functions of the lock-in amplifiers 23t and 23r are turned off, and a filter whose passband is ωs = (2ω ₂ −ω ₁ ) is used as an effective filter of the filter switching mechanisms 22t and 22r. You only have to set it.

(G) Third-order stimulated Raman loss (SRL): a nonlinear optical process in which illumination light with frequencies ω ₁ and ω ₂ is irradiated and signal light with frequency ωs = ω ₁ is detected (where ω ₁ > ω ₂ ). This SRL is represented by a double-sided Feynman diagram in FIG. Therefore, in order to use this SRL is off the pulsed laser light L1 optical frequency is omega _1, and on both the pulsed laser beam L2 optical frequency is omega _2, a modulation function of EOM126-1 and, to turn on the modulation function of EOM126-2, the lock-in amplifier 23t, and on both lock-in function of the 23r, set the filter pass band is ωs = ω ₁ filter switching mechanism 22t, the effective filter of 22r do it.

That is, in the SRL, since the optical frequency ωs of the signal light is equal to the optical frequency ω1 of the _one pulse laser light L1, in order to separate them, the other pulse laser light L2 is used as a time as shown in FIG. While modulating, the signal light whose optical frequency is ω ₁ is lock-in detected (demodulated) at the same frequency as the modulation frequency.

(H) Third-order stimulated Raman gain (SRG): a nonlinear optical process in which illumination light of frequencies ω ₁ and ω ₂ is irradiated and signal light (Raman gain) of frequency ωs = ω ₂ is detected (where ω ₁ > ω ₂ ) . This SRG is represented by a double-sided Feynman diagram in FIG. Therefore, in order to use this SRG, both the pulse laser beam L1 having the optical frequency ω ₁ and the pulse laser beam L2 having the optical frequency ω ₂ are turned on, and the modulation function of the EOM 126-1 is turned on. then, turn off the modulation function of EOM126-2, the lock-in amplifier 23t, and on both lock-in function of the 23r, set the filter pass band is ωs = ω ₂ filter switching mechanism 22t, the effective filter of 22r do it.

That is, in the SRG, since the optical frequency ωs of the signal light is equal to the optical frequency omega ₂ of one of the pulsed laser light L2, in order to separate them, and the other pulsed laser beam L1 time as shown in FIG. 3 (B) While modulating, the signal light whose optical frequency is ω ₂ is detected (demodulated) at the same frequency as the modulation frequency.

<Double-sided Feynman diagram>
Here, the double-sided Feynman diagram (FIG. 2) will be briefly described.

  A pair of parallel line segments in the double-sided Feynman diagram (FIG. 2) represents a molecular state (polarization state) that causes a nonlinear optical process. The polarization state is a state in which two states are quantum mechanically overlapped, and a pair of parallel line segments represents one state and the other state. The arrow toward the line segment represents the light absorbed by the molecule, and the arrow away from the line segment represents the light emitted from the molecule. Each time an arrow representing absorption or emission of excitation light crosses a line segment, the state transitions and the polarization state changes. The wavy arrow in the upper left represents the signal light finally emitted. As a result of emitting the signal light, the state of the molecule shifts from the polarization state to a certain final state (steady state). The symbol ω attached to the arrow means the frequency of the excitation light.

<Calculation formula stored in advance by the calculation unit 29>
Here, an arithmetic expression stored in advance by the arithmetic unit 29 will be described.

  The arithmetic expression stored in advance by the arithmetic unit 29 is the following arithmetic expression for calculating the coherent transfer function (CTF) of the nonlinear microscope.

なお、演算式中の各演算記号は、以下のとおり定義される。

Note that each calculation symbol in the calculation formula is defined as follows.

また、演算式中のＰｓ（ｆ’）は、信号光の３次元瞳関数（信号光を導光する有効対物レンズの３次元瞳関数）である。また、ｆ’は空間周波数であって、ｚ方向（光軸方向）の空間周波数ｆｚ’と、ｘ方向の空間周波数ｆｘ’と、ｙ方向の空間周波数ｆｙ’とからなる。

Further, Ps (f ′) in the arithmetic expression is a three-dimensional pupil function of the signal light (a three-dimensional pupil function of an effective objective lens that guides the signal light). Further, f ′ is a spatial frequency, and includes a spatial frequency fz ′ in the z direction (optical axis direction), a spatial frequency fx ′ in the x direction, and a spatial frequency fy ′ in the y direction.

P _R1 (f ′), P _R2 (f ′),... In the arithmetic expression are three-dimensional pupil functions (lights) of light having an optical frequency represented by an upper right solid arrow in the double-sided Feynman diagram. (A three-dimensional pupil function of an effective objective lens that guides light) (in random order).

In addition, P _L1 (f ′), P _L2 (f ′),... In the arithmetic expression are three-dimensional pupil functions (lights) of light having an optical frequency represented by a solid line arrow pointing upward in the double-sided Feynman diagram. (A three-dimensional pupil function of an effective objective lens that guides light) (in random order).

  Therefore, when the type of the nonlinear optical process is SHG (FIG. 2A), this arithmetic expression becomes the following arithmetic expression (A), and the type of the nonlinear optical process is SFG (FIG. 2B). Is the following equation (B), and when the type of nonlinear optical process is DFG (FIG. 2C), the following equation (C) is obtained, and the type of nonlinear optical process is In the case of THG (FIG. 2 (D)), the following equation (D) is obtained. When the type of the nonlinear optical process is CARS (FIG. 3 (E)), the following equation (E) is obtained. When the type of nonlinear optical process is CSRS (FIG. 3F), the following equation (F) is obtained, and when the type of nonlinear optical process is SRL (FIG. 3G): The following equation (G) is obtained, and the type of nonlinear optical process is SRG (Fig. If it is (H)) is to become the following arithmetic expression (H). Note that the subscript n in these arithmetic expressions (A) to (H) corresponds to the subscript n in the double-sided Feynman diagram.

また、或る光の３次元瞳関数Ｐ（ｆ’）は、その光を導光する有効対物レンズのＮＡと、その光の波長λと、その光の媒質の屈折率ｎ（ｎは、λに依存するので、以下、「ｎ（λ）」と表す。）とによって決まり、典型的には、値が１となる範囲と値が０となる範囲とを有した関数であって、値が１となる範囲を図示すると、図４に示すような部分球殻状となる。この部分球殻の曲率半径は、ｎ（λ）／λに一致し、部分球殻の縁から原点に向かう線分とｆｚ’軸との成す角度をθとおくと、ｓｉｎθはＮＡ／ｎ（λ）に一致する。

Further, the three-dimensional pupil function P (f ′) of a certain light includes the NA of an effective objective lens that guides the light, the wavelength λ of the light, and the refractive index n (n is λ of the light medium). Therefore, it is a function having a range in which the value is 1 and a range in which the value is 0, and the value is typically represented by “n (λ)”. When the range of 1 is illustrated, a partial spherical shell shape as shown in FIG. 4 is obtained. The radius of curvature of the partial spherical shell is equal to n (λ) / λ, and when the angle between the line segment from the edge of the partial spherical shell to the origin and the fz ′ axis is θ, sin θ is NA / n ( λ).

  Incidentally, FIG. 4 shows an example of the three-dimensional pupil function Ps (f ′) of the signal light when the illumination type is a transmission type. When the illumination type is a reflection type, the three-dimensional pupil function Ps (f ′) of the signal light is obtained by inverting the function of FIG. 4 with respect to the fx′-fy ′ plane.

<Resolution calculation processing by calculation unit 29>
Next, resolution calculation processing by the calculation unit 29 will be described.

  The resolution calculation process is executed in response to an instruction from the control unit 28. The control unit 28 determines whether or not the setting contents of the nonlinear microscope (the combination of the nonlinear optical process type, the wavelength used in the nonlinear optical process, the NA of the effective objective lens, and the illumination type) have been changed. If so, an instruction to execute the resolution calculation process is given to the arithmetic unit 29. The calculation unit 29 is provided with information on the setting contents of the nonlinear microscope (combination of the type of the nonlinear optical process, the wavelength used in the nonlinear optical process, the NA of the effective objective lens, and the illumination type) along with the execution instruction. It is done.

  When the execution instruction is given, the calculation unit 29 selects one of the calculation formulas (A) to (H) according to the type of the nonlinear optical process.

Next, the calculation unit 29 creates a three-dimensional pupil function necessary for the selected calculation formula, that is, at least one of P ₁ (f ′) and P ₂ (f ′) and Ps (f ′). The creation is performed based on the wavelength used, the NA, and the illumination type.

  Next, the calculation unit 29 calculates the CTF (f ′) of the nonlinear microscope by applying the created three-dimensional pupil function to the calculation formula.

  Next, the computing unit 29 obtains a binarized CTF by binarizing the distribution of the calculated CTF (f ′) on the fx′-fz ′ plane. This binarized CTF indicates a spatial frequency region (transmission region) in the fz ′ direction of a structure that can be transmitted from the observation target surface to the photodetector side.

  Next, the computing unit 29 displays the acquired binarized CTF on a monitor (not shown) as the resolution of the nonlinear microscope.

  FIG. 5A is a binarized CTF when the type of the nonlinear optical process is SHG, and FIG. 5B is a binarized CTF when the type of the nonlinear optical process is DFG. FIG. 5C shows the binarized CTF when the type of the nonlinear optical process is THG, and FIG. 5D shows the binary CTF when the type of the nonlinear optical process is CARS or CSRS. FIG. 5E shows the binarized CTF when the type of nonlinear optical process is SRG or SRL.

  5A to 5E, the horizontal axis is the fz ′ axis, and the vertical axis is the fx′fy ′ direction. In these FIGS. 5A to 5E, a bright area is a transmission area, and a dark area is a non-transmission area. It can be considered that the observation resolution in the z direction is higher as the width of the transmission area in the fz ′ direction is wider, and the observation resolution in the xy direction is higher as the width of the transmission area in the fx ′ direction is wider (objective lens). Since the shape is rotationally symmetric, the characteristic in the x direction is also applied to the characteristic in the y direction.)

  Each of FIGS. 5A to 5E is obtained under the following conditions.

  (1) The illumination type is a transmission type.

  (2) The NA of the effective objective lens on both sides of the observation target surface is 0.9.

  (3) The medium of the effective objective lens is air (dry type).

  (4) The difference between the optical frequencies of the two types of pulse laser beams L1 and L2 used in CARS, CSRS, SRG, and SRL is very small (the wavelength of the pulse laser beam L2 is λ).

  (5) The wavelength of the pulse laser beam L2 used in the DFG is about twice the wavelength of the pulse laser beam L1 used in the DFG (the wavelength of the pulse laser beam L2 is λ).

  Here, as shown in FIGS. 5A to 5E, when the type of the nonlinear optical process is different, the shape (that is, resolution) of the transmission region is different even if other conditions are the same. For example, when the type of the nonlinear optical process is THG, the binarized CTF at the origin is 0, so that the DC component of the structure of the observation target surface cannot be decomposed by THG. Since the binarized CTF at 1 is 1, it can be seen that the DC component of the structure of the observation target surface can be decomposed in other types.

  FIGS. 5A to 5E only show the difference in the type of the nonlinear optical process, but the combination of the wavelength used in the nonlinear optical process, the NA of the effective objective lens, and the illumination type is different. Even if the types of nonlinear optical processes are the same, the shape (that is, resolution) of the transmission band is different.

  Therefore, the user can appropriately confirm on the monitor the resolution that changes in accordance with the setting contents of the nonlinear microscope.

  Note that the calculation unit 29 may display an indication of the brightness of the signal light on the monitor together with the binarized CTF. The standard of the brightness of the signal light is determined by the susceptibility χ (3) of the nonlinear optical process. The type of the nonlinear optical process, the wavelength used in the nonlinear optical process, the NA of the effective objective lens, the illumination type, It can be calculated based on the combination with the type of the observation object. Note that the type of the observation object may be input by the control unit 28 by the user.

<Sharpening process by calculation unit 29>
Next, sharpening processing by the calculation unit 29 will be described.

  The sharpening process is executed in response to an instruction from the control unit 28. Each time the control unit 28 acquires a three-dimensional observation image (a three-dimensional reflection observation image or a three-dimensional transmission observation image), the control unit 28 gives an instruction to execute a sharpening process to the calculation unit 29. The calculation unit 29 is given the information on the CTF (f ′) calculated by the resolution calculation process together with the execution instruction.

  When the execution instruction is given, the calculation unit 29 obtains a Fourier spectrum F (f ′) by performing Fourier transform on the three-dimensional observation image I (x, y, z) (note that, as described above, f 'Consists of fx', fy ', fz').

  In addition, the calculation unit 29 calculates an autocorrelation function R (f ′) of CTF (f ′). The absolute value of the cutoff frequency of the autocorrelation function R (f ′) is larger than the absolute value of the cutoff frequency of CTF (f ′). Here, the cutoff frequency of the autocorrelation function R (f ′) is expressed as −fa ′ to fa ′.

  Next, the calculation unit 29 cuts the cut-off frequency of the Fourier spectrum F (f ′) in order to make the cut-off frequency of the Fourier spectrum F (f ′) coincide with the cut-off frequency of the autocorrelation function R (f ′). The value outside the off-frequency -fa ′ to fa ′ is replaced with zero. Hereinafter, the Fourier spectrum F (f ′) after replacement is referred to as “Fourier spectrum G (f ′)”.

  Next, the calculation unit 29 performs a frequency operation on the Fourier spectrum G (f ′) to moderate the rise of the Fourier spectrum G (f ′) (near the cutoff frequency). Hereinafter, the Fourier spectrum G (f ′) after frequency manipulation is referred to as “Fourier spectrum H (f ′)”.

  Next, the calculation unit 29 divides the Fourier spectrum H (f ′) by the autocorrelation function R (f ′), and performs inverse Fourier transform on the Fourier spectrum H (f ′) after the division, thereby performing sharpening. An observation image I (x, y, z) is acquired.

  Next, the computing unit 29 displays the sharpened observation image I (x, y, z) on a monitor (not shown).

  Therefore, the user can observe the observation image acquired by the nonlinear microscope as a clear image.

<Effect of embodiment>
As described above, the nonlinear microscope of the present embodiment condenses the illumination light supplied from the light source (laser light source 11) on the object to be observed (culture vessel 10), and causes a coherent nonlinear optical process at the condensing point. Illumination optical system (lens 121, beam splitter 122, total reflection mirror 127A, optical path length difference adjustment mechanism 128, total reflection mirrors 127B, 123, 127C, beam splitter 129, dichroic mirror 14, lenses 16A, 16B, objective lens switching mechanism 19 effective objective lenses) and a detection optical system (effective objective lens 20 of the objective lens switching mechanism 20, total reflection mirror 21, and condensing lens 25t) for detecting coherent signal light generated in the nonlinear optical process at the condensing point. , 25r, photodetectors 27t, 27r, etc.) And a calculation unit (calculation unit 29) that calculates a coherent transfer function of the microscope.

  Therefore, the nonlinear microscope of this embodiment can quantitatively evaluate the performance of the nonlinear microscope.

  Moreover, since the calculation means (calculation unit 29) stores in advance the calculation formulas (formulas (A) to (H)) for calculating the coherent transfer function, the performance evaluation of the nonlinear microscope can be performed at high speed. it can.

  The calculation means (calculation unit 29) calculates the coherent transfer function by calculating the type of nonlinear optical process, the wavelength used in the nonlinear optical process, the illumination type, the NA of the objective lens of the illumination optical system, and the objective lens of the detection optical system. Since at least one of the NAs is taken into account, the performance evaluation of the nonlinear microscope can be performed with high accuracy.

  The calculation means (calculation unit 29) includes: a type of nonlinear optical process, a wavelength used in the nonlinear optical process, an illumination type of the illumination optical system, an NA of the objective lens of the illumination optical system, and an objective lens of the detection optical system Since the calculation is performed every time at least one of the NAs is changed, it is possible to cope with a change in the setting contents of the nonlinear microscope.

  Further, since the nonlinear microscope of the present embodiment further includes a presentation unit (monitor) that presents information related to the coherent transfer function calculated by the calculation unit to the user, the user can visually recognize the performance of the nonlinear microscope. .

  The presenting means (monitor) presents the binarized image (binarized CTF) of the coherent transfer function calculated by the calculating means (calculating unit 29) to the user, so that the user can intuitively understand the performance of the nonlinear microscope. Can be recognized.

  In addition, the nonlinear microscope of the present embodiment is a control means (stage 100, optical scanner) that acquires an image of the surface to be observed by repeatedly detecting the signal light while scanning the surface to be observed of the object to be observed with a condensing point. 15) is further provided, so that an image of the object to be observed (transmission observation image or reflection observation image) can be obtained with high resolution.

  In addition, since the nonlinear microscope of the present embodiment further includes sharpening means (calculation unit 29) that sharpens the image of the surface to be observed based on the coherent transfer function, the coherent transfer function can be effectively used.

  In the nonlinear microscope of this embodiment, the types of nonlinear optical processes are the first harmonic (SHG), the sum frequency (SFG), the difference frequency (DFG), the second high frequency (THG), the coherent anti-Stokes Raman scattering ( Since it is possible to switch between at least two of CARS), coherent Stokes Raman scattering (CSRS), third-order stimulated Raman loss (SRL), and third-order stimulated Raman gain (SRG), the same by a plurality of different types of nonlinear optical processes The observed surface can be observed.

[Supplement to the first embodiment]
In addition, although the calculating part 29 of 1st Embodiment displayed the binarized CTF in the fx'-fz 'plane on the monitor, it cannot be overemphasized that the binarized CTF in another plane may be displayed on a monitor. . Alternatively, binarized CTFs in a plurality of planes may be displayed on the monitor.

  Moreover, although the calculating part 29 of 1st Embodiment displayed the binarized CTF on the monitor, you may display the CTF before binarization on a monitor. In this case, the CTF value distribution may be expressed by a gradation distribution, a hue distribution, or the like.

  The nonlinear microscope of the first embodiment includes confocal pinholes (pinhole masks 26t and 26r), but may be omitted. However, in the sharpening process in that case, it is desirable to use the following function H (f ') instead of using CTF (f').

  This function H (f ′) is a function calculated by an arithmetic expression in which Ps (f ′) is replaced with a delta function δ (f ′) in the arithmetic expression for calculating CTF (f ′).

  In addition, some or all of the functions of the calculation unit 29 of the first embodiment may be mounted as hardware in the nonlinear microscope or may be mounted as software of the nonlinear microscope. When implemented as software, the software may be installed in a computer of a nonlinear microscope via the Internet or a storage medium.

  The coherent transfer function (CTF) described in the first embodiment is defined as follows.

なお、この式における演算記号Fは、フーリエ変換を意味する。

Note that the operation symbol F in this equation means Fourier transform.

  In the first embodiment, when the illumination type of the nonlinear microscope is set to the reflection type, the nonlinear microscope is operated as the laser scan type. However, the nonlinear microscope may be operated as a stage scan type. In that case, the optical scanner 15 is unnecessary.

  DESCRIPTION OF SYMBOLS 11 ... Laser light source, 122, 129 ... Beam splitter, 125-1, 125-2 ... Optical parametric oscillator (OPO), 126-1, 126-2 ... Electro-optic modulator (EOM), 14 ... Dichroic mirror, 15 ... Optical scanner, 16A ... lens, 16B ... lens 16, 19, 20 ... objective lens switching mechanism, 100 ... stage, 22t, 22r ... filter switching mechanism, 25t, 25t ... condensing lens, 26r, 26t ... pinhole mask, 27r , 27t ... photodetector, 23t, 23r ... lock-in amplifier, 28 ... control unit, 29 ... calculation unit

Claims (18)

An illumination optical system that condenses the illumination light supplied from the light source on the object to be observed and causes a coherent nonlinear optical process at the condensing point;
A non-linear microscope comprising: a detection optical system that detects coherent signal light generated in the non-linear optical process at the condensing point;
A non-linear microscope, comprising: a calculating unit that calculates a coherent transfer function of the non-linear microscope. The nonlinear microscope according to claim 1,
The calculating means includes
A non-linear microscope, wherein an arithmetic expression for calculating the coherent transfer function is stored in advance. The nonlinear microscope according to claim 1 or 2,
The calculating means includes
A nonlinear microscope characterized in that the type of the nonlinear optical process is added to the calculation of the coherent transfer function. In the nonlinear microscope according to any one of claims 1 to 3,
The calculating means includes
A nonlinear microscope, wherein a wavelength used for the nonlinear optical process is added to the calculation of the coherent transfer function. In the nonlinear microscope according to any one of claims 1 to 4,
The calculating means includes
A nonlinear microscope, wherein an illumination type of the illumination optical system is added to the calculation of the coherent transfer function. In the nonlinear microscope according to any one of claims 1 to 5,
The calculating means includes
A nonlinear microscope, wherein the NA of the objective lens of the illumination optical system is added to the calculation of the coherent transfer function. In the nonlinear microscope according to any one of claims 1 to 6,
The calculating means includes
A nonlinear microscope, wherein the NA of the objective lens of the detection optical system is added to the calculation of the coherent transfer function. The nonlinear microscope according to claim 3,
The type of the nonlinear optical process can be changed,
The calculating means includes
The nonlinear microscope, wherein the calculation is performed every time the type of the nonlinear optical process is changed. The nonlinear microscope according to claim 4,
The wavelength used in the nonlinear optical process can be changed,
The calculating means includes
The nonlinear microscope characterized in that the calculation is performed every time the wavelength used in the nonlinear optical process is changed. The nonlinear microscope according to claim 5,
The illumination type of the illumination optical system can be switched between a transmission type and a reflection type,
The calculating means includes
The nonlinear microscope, wherein the calculation is performed every time the illumination type of the illumination optical system is switched. The nonlinear microscope according to claim 6,
The NA of the objective lens of the illumination optical system can be changed,
The calculating means includes
The nonlinear microscope, wherein the calculation is performed every time the NA of the objective lens is changed. The nonlinear microscope according to claim 7,
The NA of the objective lens of the detection optical system can be changed,
The calculating means includes
The nonlinear microscope, wherein the calculation is performed every time the NA of the objective lens is changed. In the nonlinear microscope according to any one of claims 1 to 12,
A non-linear microscope, further comprising: presentation means for presenting information about the coherent transfer function calculated by the calculation means to a user. The nonlinear microscope according to claim 13,
The presenting means is
A non-linear microscope, wherein the binarized image of the coherent transfer function calculated by the calculating means is presented to the user. In the nonlinear microscope according to any one of claims 1 to 14,
A non-linear microscope, further comprising control means for acquiring an image of the surface to be observed by repeatedly detecting the signal light while scanning the surface to be observed of the object to be observed at the condensing point. The nonlinear microscope according to claim 15, wherein
A nonlinear microscope, further comprising sharpening means for sharpening an image of the surface to be observed based on the coherent transfer function. The nonlinear microscope according to claim 8, wherein
The type of nonlinear optical process is:
First harmonic (SHG), sum frequency (SFG), difference frequency (DFG), second high frequency (THG), coherent anti-Stokes Raman scattering (CARS), coherent Stokes Raman scattering (CSRS), third-order stimulated Raman loss (SRL) ), A nonlinear microscope characterized in that it can be switched between at least two of the third-order stimulated Raman gains (SRGs). An illumination procedure for condensing illumination light supplied from a light source on an object to be observed and causing a coherent nonlinear optical process at the condensing point;
A non-linear observation method including a detection procedure for detecting coherent signal light generated in the non-linear optical process at the condensing point,
The nonlinear observation method characterized by including the calculation procedure which calculates the coherent transfer function of the said nonlinear observation method.

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