JP2018205069A - Optical measuring device - Google Patents

Abstract

An optical measuring device capable of correcting a defocus due to correcting spherical aberration and ensuring signal intensity is provided.
An optical measurement apparatus according to the present invention describes a positional relationship between an aberration correction lens and an objective lens that can correct spherical aberration while suppressing the focal position of the combined light within the sample within a predetermined range. The stored data is stored in advance, and the spherical aberration is corrected according to the data.
[Selection] Figure 6

Description

  The present invention relates to an optical measurement device that performs measurement by irradiating a sample with light.

  The optical microscope has been developed as an indispensable apparatus in the natural science, engineering, and industrial fields. In particular, in recent years, an optical microscope is sometimes used when observing cells in the fields of regenerative medicine and drug discovery, and there is an increasing need for enhancing the function of the optical microscope. In current cell analysis, cells are generally stained with a reagent and then observed with a microscope or the like. However, since this method has an effect on cells due to staining, it is difficult to continuously analyze the same cells and to directly use the examined cells for medical purposes.

  A CARS (Coherent Anti-Stokes Raman Scattering) microscope can perform molecular identification at a higher speed than a Raman microscope by applying a nonlinear optical effect. Since the CARS microscope can observe a sample non-invasively, it is suitable for cell observation.

  The following Patent Document 1 describes a configuration example in which only a specific spectral component is detected from CARS light. The following Patent Document 2 describes a multicolor CARS microscope that spectrally detects CARS light generated using a broadband light source. Since the multicolor CARS microscope can estimate the Raman spectrum from the spectrum of CARS light, the amount of information that can be acquired is large, and the measurement object can be analyzed in more detail.

  Patent Document 3 below discloses a method for identifying and correcting a spherical error when a sample is imaged with a microscope. In this document, the control unit corrects the spherical error by electrically adjusting the correction lens element. Furthermore, the defocus situation which arises during a microscopic inspection is eliminated through an autofocus control apparatus.

US Pat. No. 6,108,081 JP 2009-222531 A JP2013-088809A

  Patent Document 3 described above is a technique for performing spherical aberration correction and autofocus using reflected light, and is considered effective when observing a reflecting surface (for example, the surface of a sample). On the other hand, when a sample having no reflective surface is measured by CARS, such as when observing the inside of a cell, a new problem arises.

  When imaging is performed by moving a cell along the optical axis direction in CARS, the sample thickness between the condensing objective lens and the condensing position is changed by the movement, thereby generating spherical aberration. In order to ensure the signal intensity, dynamic spherical aberration correction corresponding to the position of the condenser objective lens is effective. However, when the spherical aberration correction is performed on the other side, the condensing position is changed by the spherical aberration correction. Accordingly, the CARS light generation point is shifted from the focal position of the detection objective lens, and the signal intensity is reduced.

  The present invention has been made in view of the above-described problems, and an object thereof is to provide an optical measurement device capable of correcting a defocus due to correcting spherical aberration and ensuring signal intensity. To do.

  The optical measurement device according to the present invention can correct spherical aberration while suppressing the focal position of the combined light within the sample within a predetermined range, and describes data describing the positional relationship between the aberration correction lens and the objective lens. The spherical aberration is corrected in accordance with the data stored in advance.

  According to the optical measurement apparatus of the present invention, even when the inside of a sample is measured by CARS, the signal intensity is prevented from being reduced by correcting the spherical aberration, and the measurable depth is improved. be able to.

It is an energy level diagram in CARS. One process related to the non-resonant term χ _nr ⁽³⁾ is shown. It is an example of a process in the case of using a broadband light source. It is a block diagram of the conventional multicolor CARS microscope. It is a schematic diagram which shows the structural example of the optical measuring device 1000 which concerns on Embodiment 1. FIG. It is a schematic diagram showing details of aberration correction by the aberration correction mechanism 750. It is a calculation result showing the relationship between the movement amount dz1 of the aberration correction lens 752 and the movement amount dz2 of the objective lens 713 for detecting high-intensity CARS light while keeping the focal position inside the sample 714 constant. It is a calculation result which shows the relationship between the measurement depth in the sample 714 inside, and the cube of the spot light intensity. The calculation result of the moving amount dz1 of the aberration correction lens 752 used when performing the calculation shown in FIG. The calculation result of the movement amount dz2 of the objective lens 713 used when performing the calculation shown in FIG. 8 is shown. The change of the focus position in the calculation result shown in FIG. 8 is shown. The result of calculating dz1 (z, n0) according to Equation 3 is shown. The result of calculating dz2 (z, n0) according to Equation 4 is shown. A calculation result of the spot light intensity in the case where aberration correction is performed by simultaneously moving the aberration correction lens 752 and the objective lens 713 according to Expressions 1 and 2 is shown. A change in the light spot position when aberration correction is performed by simultaneously moving the aberration correction lens 752 and the objective lens 713 according to Expressions 1 and 2 is shown. 10 is a schematic diagram illustrating details of aberration correction by an aberration correction mechanism 750 according to Embodiment 3. FIG. A mode that the measurement depth in a sample is made into a desired value is shown. A state in which dz1 is controlled is shown. The relationship between the wavefront aberration and the cube of the spot light intensity is shown. It is a flowchart which shows the control procedure by the controller 700 in Embodiment 1-3. 6 is a schematic diagram of an aberration correction mechanism 750 according to Embodiment 4. FIG. The calculation result which shows the phase distribution which the spatial phase modulator 753 provides is shown. It is a calculation result which shows the relationship between the measurement depth inside the sample 714, and the cube of the spot light intensity. It is a block diagram of the optical measuring device 1000 which concerns on Embodiment 5. FIG. 10 is a flowchart for explaining the operation of the optical measurement apparatus 1000 according to the fifth embodiment. It is a block diagram of the optical measuring device 1000 which concerns on Embodiment 6. FIG. It is a modification of Embodiment 6. 14 is a flowchart for explaining the operation of the optical measurement apparatus 1000 according to the sixth embodiment. It is the result of having compared the CARS spectrum of an adipocyte before and after the chromatic aberration correction demonstrated in this Embodiment 6. FIG. 10 is a schematic diagram illustrating an aberration correction mechanism 750 according to Embodiment 7. FIG. 10 is a schematic diagram illustrating another configuration example of an aberration correction mechanism 750 according to Embodiment 7. FIG. The calculation result of the movement amount for re-correcting the position of the objective lens 713 is shown.

<Basic principle of CARS microscope>
In order to facilitate understanding of the present invention, the outline of the basic principle of the CARS microscope and the problems associated with correcting spherical aberration will be described first. After that, an embodiment of the present invention will be described.

CARS is light emission by third-order polarization. In order to generate CARS light, pump light, Stokes light, and probe light are required. Generally, in order to reduce the number of light sources, the probe light is substituted with pump light. In this case, the induced third-order polarization is expressed by the following formula A. χ _r ⁽³⁾ (ω _AS ) is a resonance term of molecular vibration of third-order electrical susceptibility, and χ _nr ^{(3) having} no frequency dependence is a non-resonance term. Represents an electric field of the pump light and the probe light E _P, the electric field of the Stokes light is expressed by E _S. An asterisk attached to the shoulder of the E _S denotes the complex conjugate. The intensity of the CARS light is represented by the following formula B.

P _AS ⁽³⁾ (ω _AS ) = | χ _r ⁽³⁾ (ω _AS ) + χ _nr ⁽³⁾ | E _P ² (ω _P ) E ^* _S (ω _S ) (A)
I _CARS (ω _AS ) ∝ | P _AS ⁽³⁾ (ω _AS ) | ² (B)

FIG. 1 is an energy level diagram in CARS. The mechanism by which CARS light is generated will be described using the energy level diagram of the molecule shown in FIG. FIG. 1 illustrates the resonance term process of Equation 1. Reference numeral 2001 represents a ground state of the molecule, and reference numeral 2002 represents a vibrationally excited state. Simultaneously irradiating the Stokes beam frequency omega _P pump light and the frequency omega _S of. At this time, the molecule is excited to one of the vibrationally excited states 2002 through the intermediate state 2003. It is irradiated with probe light frequency omega _P in a molecule in an excited state, while through the intermediate state 2004 generates CARS light frequency omega _AS, molecules returns to the ground state 2001. The frequency of the CARS light at this time is expressed as ω _AS = 2 · ω _P −ω _S.

FIG. 2 shows one process related to the non-resonant term χ _nr ⁽³⁾ among the third-order polarizations. In the process of Figure 2, by simultaneous irradiation with Stokes beam frequency omega pump light _P and the frequency omega _'S, intermediate state 2005 rather than the vibrational excitation status 2002 is excited. By irradiating a probe light of a frequency omega _P, CARS light nonresonant frequency omega _AS via an intermediate state 2004 occurs.

  FIG. 3 is an example process when using a broadband light source. Among CARS microscopes, those that spectrally detect CARS light generated by using a broadband light source as Stokes light are called multicolor CARS microscopes (or multiplex CARS microscopes).

  FIG. 4 is a configuration diagram of a conventional multicolor CARS microscope. The output from the short pulse laser light source 2301 is branched into two by a beam splitter 2302. One of the branched lights is introduced into an optical fiber such as the photonic crystal fiber 2303, and broadband light (referred to as supercontinuum light) is generated therein. When the supercontinuum light is emitted from the optical fiber, the long pass filter 2304 extracts only a desired wavelength component (a component having a wavelength longer than that of the excitation light), and this is used as Stokes light. The other excitation light and Stokes light are combined by a dichroic mirror 2305. The combined light is condensed and irradiated onto the sample 2306 by the condenser objective lens 2309. Thereby, CARS light is generated from the sample 2306, and the detection objective lens 2310 collects the CARS light and converts it into parallel light. The spectrometer 2307 acquires the wavelength spectrum by detecting the CARS light.

  The stage 2308 moves the position of the sample 2306 to acquire a CARS spectrum at each spatial point in the sample 2306. By mapping the spectral intensity of a specific wavelength to a position, a CARS spectral image is obtained.

  As described above, when the CARS spectrum image is obtained, it is general that the position of the sample 2306 is moved instead of scanning the incident light. This is because it is easy to make the focal position of the condenser objective lens 2309 coincide with the focal position of the detection objective lens 2310.

  In the CARS microscope, a lens having a high NA (numerical aperture) (for example, about 0.9) is used as the condenser objective lens 2309 in order to ensure power density. In this case, spherical aberration occurs in which the condensing position of light passing near the center of the lens and the condensing position of light passing through the lens periphery are shifted, and thus the spherical aberration is corrected by an aberration correction lens or the like. However, by correcting the spherical aberration, the focal position of the condenser objective lens 2309 is deviated from the focal position of the detection objective lens 2310, so that there is a problem that the light intensity at the time of detection is lowered.

<Embodiment 1>
FIG. 5 is a schematic diagram illustrating a configuration example of the optical measurement device 1000 according to the first embodiment of the present invention. The optical measurement apparatus 1000 includes a beam expander type aberration correction mechanism 750. The aberration correction mechanism 750 operates in accordance with an instruction from the controller 700. The controller 700 includes an input interface (for example, an interface 1110 described later) that receives measurement instructions from the user and an output interface that displays measurement results while controlling the overall operation of the optical measurement apparatus 1000.

  The short pulse laser light source 701 emits short pulse laser light in accordance with an instruction from the controller 700. The short pulse laser light source 701 is a light source such as a titanium sapphire laser, a fiber laser, or a microchip laser, and has a pulse width of nanoseconds or less. The peak power is preferably on the order of kilowatts or more because it induces a nonlinear optical effect. The wavelength may be appropriately selected based on the absorption band to be measured, the wavelength specification of the optical component used, and the like. For example, wavelengths such as 800 nm and 1064 nm can be used.

  The laser light is incident on the half-wave plate 702 and the polarization beam splitter 703. The half-wave plate 702 changes the polarization direction of the laser light in accordance with an instruction from the controller 700. The polarization beam splitter 703 branches the laser light into a transmission component and a reflection component with a power branching ratio based on the polarization direction.

  The laser light transmitted through the polarization beam splitter 703 is condensed on the end face of the photonic crystal fiber 705 by the condenser lens 704. The photonic crystal fiber 705 is an optical fiber in which a honeycomb-like hollow clad is formed around the core, and the incident light is strongly confined in the core. When a short pulse laser beam is incident on the photonic crystal fiber 705, nonlinear optical phenomena such as self-phase modulation and four-wave mixing are induced, thereby generating supercontinuum light having a wide spectrum.

  The generated supercontinuum light becomes parallel light by the collimating lens 706, and after the short wavelength component is cut by the long pass filter 707, the supercontinuum light reaches the dichroic mirror 708. The dichroic mirror 708 reflects the pump light wavelength and transmits light of other wavelengths. The aberration correction mechanism 750 performs wavefront correction on the transmitted light according to the type of sample 714 and the measurement depth. The corrected light enters the objective lens 713 as Stokes light.

  The laser light reflected by the polarization beam splitter 703 is reflected by the mirror 709, the mirror 712, and the dichroic mirror 708, and similarly subjected to wavefront correction by the aberration correction mechanism 750, and then enters the objective lens 713 as pump light. To do.

  The objective lens 713 collects the broadband Stokes light and the pump light, which are coaxially combined by the dichroic mirror 708, with respect to the sample 714. In order to increase the energy density of Stokes light and pump light in the sample 714 and improve the efficiency of generating CARS light, it is desirable that the numerical aperture of the objective lens 713 is as high as 0.8 or more, for example.

  The CARS process described above is induced in the sample 714, and CARS light having a wavelength corresponding to the molecular species of the sample 714 is generated. The collimating lens 716 converts CARS light into parallel light. The notch filter 717 and the short pass filter 718 cut the transmitted component of pump light and Stokes light. The spectroscope 719 detects the CARS spectrum and notifies the controller 700 of the result.

  The aberration correction mechanism 750 has a set of aberration correction lenses that share a focal point. In accordance with an instruction from the controller 700, the aberration correction mechanism 750 uses one of the aberration correction lenses in the optical axis direction using a drive mechanism (not shown) according to the type (refractive index) and measurement depth of the sample 714. The spherical aberration is corrected by moving along (z direction). At this time, the controller 700 moves the objective lens 713 along the optical axis direction using a drive mechanism (not shown) according to a predetermined relationship (details will be described later) so that the focal position inside the sample 714 does not change. . As described above, even if the type and measurement depth of the sample 714 are changed, the attenuation of the generated CARS light is suppressed, and the attenuation of the CARS light received by the spectroscope 719 is suppressed.

  FIG. 6 is a schematic diagram showing details of aberration correction by the aberration correction mechanism 750. Here, a configuration example using the aberration correction lenses 751 and 752 as the aberration correction mechanism 750 is shown. These lenses constitute a beam expander optical system sharing a focal point. The storage unit 1100 is a storage device that stores a data table described later.

  The aberration correction lens 752 and the objective lens 713 are mounted on actuators 761 and 762, respectively. The actuators 761 and 762 independently control the positions of the aberration correction lenses 751 and 752 along the optical axis direction (z direction) according to instructions from the controller 700.

  When observing a depth position of, for example, 100 μm or more inside the sample 714, spherical aberration occurs according to the refractive index of the sample 714. As a result, as shown in FIG. 6, the focal position changes according to the aperture radius position of the objective lens 713. Since the CARS light is generated in proportion to the cube of the light intensity of the spot, if the focal position shifts, the CARS light is greatly attenuated and it becomes difficult to correctly acquire the information of the sample 714. The amount of movement of the aberration correction lens is dz1, the amount of movement of the objective lens is dz2, and the controller 700 appropriately determines dz1 and dz2, thereby correcting the spherical aberration while keeping the focal position inside the sample 714 constant. Can do.

  Hereinafter, the effect of the present invention will be verified by calculating the wavefront aberration and the spot light intensity using the ray tracing method. Unless otherwise noted, the optical conditions are as follows: the wavelength of the pump light is 1064 nm, the objective lens 713 has a water immersion type numerical aperture of 1.2, and the aberration correction lens 752 has a numerical aperture of 0.15.

  The refractive index of water is 1.333, and the refractive index of a general cover glass is 1.52. According to publicly known literature (Biomedical Photonics Handbook 2nd Edition, volume 1, CRC Press, Tuan Vo-Dinh.), The refractive index of human tissue is the brain, heart, 1.36 to 1.38 in the main tissues such as kidney, spleen, stomach, and muscle, and 1.39 to 1.40 in the bladder wall and artery. Since cells such as iPS cells and ES cells are cultured in a culture solution, the refractive index is close to that of water, and is considered to be about 1.333 to 1.35 depending on the density of the cells and the state of the culture solution. Can do.

  FIG. 7 is a calculation showing the relationship between the movement amount dz1 of the aberration correction lens 752 and the movement amount dz2 of the objective lens 713 for detecting high intensity CARS light while keeping the focal position inside the sample 714 constant. It is a result. The refractive index of the sample 714 was 1.38. By controlling the positions of the aberration correction lens 752 and the objective lens 713 in the optical axis direction according to the relationship shown in FIG. 7, the wavefront aberration can be minimized without changing the focal position. When the refractive index of the sample 714 changes, the relationship of FIG. 7 also changes. However, as will be described later, simple first-order approximation correction can be applied in the refractive index range of the living tissue.

  FIG. 8 is a calculation result showing the relationship between the measurement depth inside the sample 714 and the cube of the spot light intensity. Since the intensity of the CARS light is proportional to the cube of the spot light intensity, the vertical axis in FIG. 8 is proportional to the intensity of the CARS light.

  FIG. 8A shows the result when aberration correction is not performed. When the refractive index of the sample 714 is 1.36 to 1.38 (corresponding to the main body tissue), it can be seen that the intensity of the CARS light is attenuated to ½ or less at a depth of about 50 μm.

  FIG. 8B shows a calculation result when aberration correction is performed by moving the aberration correction lens 752 and the objective lens 713 simultaneously according to the relationship shown in FIG. It can be seen that when the refractive index of the sample 714 is in the range of 1.34 to 1.39, the CARS light intensity can be maintained at ½ or more over a depth of 500 μm or more. When the cell concentration is low as in the case of cultured cells, the refractive index is in the range of 1.333 to 1.34, so it goes without saying that the measurement depth can be further increased. When the refractive index of the sample 714 is 1.40, the range in which the CARS light intensity can be maintained at ½ or more is a depth of about 400 μm. A refractive index of 1.525 corresponds to the case of a general cover glass, and it can be seen that aberration correction can be made only to a depth of about 100 μm.

  FIG. 9 shows a calculation result of the movement amount dz1 of the aberration correction lens 752 used when performing the calculation shown in FIG. As can be seen from FIG. 9, dz1 increases as the refractive index of the sample 714 increases.

  FIG. 10 shows a calculation result of the movement amount dz2 of the objective lens 713 used when performing the calculation shown in FIG. As shown in FIG. 10, it can be seen that the absolute value of dz2 increases as the refractive index of the sample 714 increases.

  FIG. 11 shows a change in the focal position in the calculation result shown in FIG. As shown in FIG. 11, it can be seen that the focal position does not change regardless of the measurement depth inside the sample 714. The range of refractive index of sample 714 is 1.333 to 1.40.

<Embodiment 1: Summary>
The optical measurement apparatus 1000 according to the first embodiment corrects the aberration according to the refractive index of the sample 714 by controlling the position of the aberration correction lens 752 and the position of the objective lens 713 according to the relationship shown in FIG. be able to. Thereby, high-intensity CARS light can be acquired at a measurement depth of 100 μm or more.

  Some objective lenses used in conventional microscopes include an aberration correction ring that responds to changes in the thickness of the cover glass. In such an objective lens, it is inevitable that the focal position changes due to aberration correction. In addition, the refractive index of a standardized inorganic cover glass is constant, and it is difficult to measure while maintaining a large CARS light intensity inside a living tissue where the refractive index changes depending on the type and culture state of cells and tissues. It is. According to the first embodiment, even such a sample is advantageous in that the CARS light intensity can be maintained large.

<Embodiment 2>
Unlike the case of a cover glass which is an inorganic substance, in a biological sample, the refractive index varies in a continuous numerical value depending on the individual sample or conditions such as the number of days of culture. If the refractive index changes even for the same sample, it can be regarded as a different sample. In the second embodiment of the present invention, a method for correcting aberration according to the refractive index change of such a biological sample will be described. The configuration of the optical measurement device 100 is the same as that of the first embodiment.

The position control amount dz1 of the aberration correction lens 752 and the position control amount dz2 of the objective lens 713 are functions dz1 (z, n) and dz2 (z, z) of the measurement depth z inside the sample 714 and the refractive index n of the sample 714. ). These two functions are obtained in advance for the representative refractive index n0 (the refractive index of a certain sample). If the refractive index of the surrounding medium of the objective lens 713 (here, water is assumed) is n _WATER , these functions dz1 (z, n) and dz2 (z, n) for a new sample having the refractive index n are It can be approximated by the following equations 1 and 2. For example, when n0 = 1.38, dz1 (z, n0) and dz2 (z, n0) have the relationship shown in FIG.

  In the following, in order to verify whether the approximations by the equations 1 and 2 are valid, the following equations 3 and 4 obtained by modifying the equations 1 and 2 are used, and dz1 (z, n0) and dz2 (z, n0) is actually calculated.

  FIG. 12 shows the result of calculating dz1 (z, n0) according to Equation 3. In FIG. 12, n0 = 1.38 and the calculation result of dz1 (z, n) shown in FIG. 9 is used. As shown in FIG. 12, it can be seen that the characteristics are almost uniform in the range of n = 1.34 to 1.40 (corresponding to the refractive index of the living body). In other words, it can be understood that the aberration can be corrected according to an arbitrary refractive index of the biological sample by approximation of Equation 1. The refractive index n = 1.525 corresponds to a general cover glass, and cannot be approximated because the characteristic separation is large. In other words, the technique used when correcting the thickness of the conventional cover glass corresponds to the curve of refractive index n = 1.525 in FIG. 12, and is different from the aberration correction method in the present invention. It can be said.

  FIG. 13 shows the result of calculating dz2 (z, n0) according to Equation 4. In FIG. 13, n0 = 1.38 and the calculation result of dz2 (z, n) shown in FIG. 10 is used. As shown in FIG. 13, it can be seen that the characteristics are almost uniform in the range of n = 1.34 to 1.40 (corresponding to the refractive index of the living body). Further, it can be seen that the refractive index n = 1.525 cannot be approximated because the separation of characteristics is large as in FIG.

  As described above, it has been shown that approximation of Expression 1 and Expression 2 is effective in the range of the refractive index of 1.34 to 1.40. When the cell density is low as in the case of cultured cells in the culture solution, the refractive index is considered to be in the range of 1.333 to 1.34, and the aberration correction amount is very small. Needless to say, the approximation of Equation 1 and Equation 2 holds.

  FIG. 14 shows the calculation result of the spot light intensity when the aberration correction is performed by moving the aberration correction lens 752 and the objective lens 713 simultaneously according to the expressions 1 and 2. The horizontal axis represents the measured depth in the sample, and the vertical axis represents the cube of the spot light intensity. As shown in FIG. 14, it can be seen that the attenuation of the cube of the spot light intensity when this approximation method is used is less than 10% in the range of the refractive index of 1.34 to 1.40. On the other hand, when the refractive index is 1.525 corresponding to a general cover glass, the attenuation of the CARS light intensity is large.

  FIG. 15 shows changes in the light spot position when aberration correction is performed by simultaneously moving the aberration correction lens 752 and the objective lens 713 in accordance with Expressions 1 and 2. In the range of the refractive index of 1.34 to 1.40, the change of the focal position is within ± 1 μm. Since the typical size of the cell is 10 μm, it can be seen that the focal position can be controlled with sufficient accuracy. On the other hand, when the refractive index is 1.525 corresponding to a general cover glass, the shift of the focal position is large.

<Embodiment 3>
FIG. 16 is a schematic diagram illustrating details of aberration correction by the aberration correction mechanism 750 according to the third embodiment of the present invention. The controller 700 controls the position of the sample 714 by the actuator 763 so that the focal position of the objective lens 713 becomes the boundary surface of the sample 714. The controller 700 uses the actuators 761 and 762 to control the aberration correction lens 752 and the objective lens 713 to the origin position where the aberration correction amount becomes zero. Since the other configuration is the same as that of the first embodiment, the difference will be mainly described below.

  When the pump light is irradiated onto the sample 714, the reflected light from the sample 714 boundary passes through the objective lens 713 and the aberration correction mechanism 750 and is reflected by the dichroic mirror 708. The reflected light passes through the dichroic mirror 770, is collected by the lens 773, and is received by the photodetector 774. By adjusting the position of the sample 714 so that the amount of light detected by the photodetector 774 is maximized, the position of the boundary surface of the sample 714 can be determined. The position in the optical axis direction of the actuator 763 at this time is set to dz3 = 0.

  FIG. 17 shows how the measured depth in the sample becomes a desired value. The position of the actuator 763 at this time is dz3 = z. As described above, at this time, the focal position shifts according to the opening position of the objective lens 713 due to the aberration according to the refractive index of the sample 714.

  FIG. 18 shows how dz1 is controlled. According to the relationship shown in FIG. 7, the position dz1 of the aberration correction lens 752 is controlled by the actuator 761, and at the same time, the position dz2 of the objective lens 713 is controlled by the actuator 762. Thereby, spherical aberration can be corrected while adjusting the focal position of the objective lens 713 to a desired position.

  FIG. 19 shows the relationship between the wavefront aberration and the cube of the spot light intensity. The CARS light generated at the focal point passes through the objective lens 713 and the aberration correction mechanism 750, is reflected by the dichroic mirror 708, is reflected by the dichroic mirror 770, is condensed by the lens 771, and is received by the photodetector 772. FIG. 19A shows the relationship between the position dz1 of the aberration correction lens 752 and the wavefront aberration at this time, and FIG. 19B shows the relationship between dz1 and the cube of the spot light intensity.

  FIG. 19 shows the calculation results of the wavefront aberration and the cube of the spot light intensity under each condition of z = 100, 200, 300, 400, and 500 μm. The refractive index n0 of the sample 714 is 1.38. For example, when z = 200 μm, when dz1 = 665 μm, the wavefront aberration is minimum, the cube of the spot light intensity is maximum, and the generated CARS light quantity is maximum. Since dz1 (z, n0) is to correct the aberration and make the focal point of the objective lens 713 coincide with the measurement depth, the movement amount of the aberration correction lens 752 at this time is dz1 (z, n0). become.

  Similarly, for another sample having a refractive index n, when the CARS light intensity detected by the photodetector 772 is maximized, the movement amount of the aberration correction lens 751 is dz1 (z, n). The refractive index n can be obtained by the following formula 5 obtained by modifying the formula 1. In Expression 5, the right side is a known value dz1 (z, n0) and dz1 (z, n) when the light intensity is maximum, and the left side is a known value except for the refractive index n. Therefore, it is clear that the refractive index n can be obtained by calculation using Equation 5.

  Replace the left side of Equation 5 with α. Since the right side of Equation 1 and the right side of Equation 2 also include α, by using α obtained as an actual measurement result according to the third embodiment, dz1 is changed every time the measurement depth inside the sample 714 is changed. There is no need to adjust dz2. That is, dz1 and dz2 can be obtained by calculation according to Equation 1, Equation 2, and α at an arbitrary measurement depth inside the sample 714.

  FIG. 20 is a flowchart illustrating a control procedure by the controller 700 in the first to third embodiments. In step S011, the controller 700 detects the boundary position of the sample 714. In step S012, the controller 700 moves the sample 714 to the measurement depth. In step S013, the controller 700 obtains the positional relationship that maximizes the CARS light amount while moving the position of the aberration correction mechanism 750 and the position of the objective lens 713 according to FIG. In step S014, the controller 700 generates a table describing the movement amount dz1 of the aberration correction mechanism 750 and the movement amount dz2 of the objective lens 713 at an arbitrary measurement depth inside the sample 714 based on the obtained positional relationship. In step S015, the controller 700 performs measurement at the specified measurement depth inside the sample 714 while controlling the position of the aberration correction mechanism 750 and the position of the objective lens 713 according to the generated table.

  In the present embodiment, for simplification of description, the sample 714 has a uniform refractive index in the depth direction. On the other hand, there are many samples such as actual living tissue and cell sheets having a layered structure in the measurement depth range. In this case, the conditions for maximizing the CARS light amount described in FIGS. 16 to 18 are implemented at each measurement depth, and correspond to the average refractive index of the medium until the pump light and Stokes light reach the focal point. Find the controlled variable. About arbitrary measurement depth inside sample 714, a control amount can be defined by interpolating them.

  In the third embodiment, a method for correcting aberration using the photodetectors 772 and 774 has been described. In the third embodiment, it is also effective to arrange a pinhole of an appropriate size in front of the photodetectors 772 and 774 in order to improve the detection accuracy with respect to the focal position.

  In Embodiments 1 to 3, the representative refractive index is n0 = 1.38. However, as shown in FIGS. 12 and 13, as shown in FIGS. It goes without saying that the control accuracy is improved by selecting the refractive index as the representative refractive index.

  In the third embodiment, without using the photodetectors 772 and 774, it is possible to detect the boundary of the sample 714 and obtain the condition that maximizes the CARS light quantity. In the optical measurement apparatus 1000 shown in FIG. 1, a microscope optical system is added to the optical system of the detection objective lens 715, and the contrast of the microscope image at the boundary of the sample 714 is maximized by a general method such as a phase difference method. The boundary position of the sample 714 can be determined by adjusting the position of the actuator 763. Thereby, the function of the photodetector 774 can be substituted. After moving the sample 714 to a predetermined measurement depth, the position of the aberration correction lens 752 and the position of the objective lens 713 are controlled so that the amount of light received by the spectroscope 719 is maximized. Functions can be substituted.

  In the above embodiment, as described with reference to FIGS. 9 and 10, dz1 and dz2 are obtained in advance for each combination of measurement depth and refractive index, and stored in the storage unit 1100 as a data table. It is also possible to automatically control dz1 and dz2 using a data table corresponding to the refractive index n obtained according to the above.

<Embodiment 4>
FIG. 21 is a schematic diagram of an aberration correction mechanism 750 according to Embodiment 4 of the present invention. In the fourth embodiment, the aberration correction mechanism 750 includes a spatial phase modulator (SLM) 753 in addition to the aberration correction lenses 751 and 752. The spatial phase modulator 753 is an optical element capable of arbitrary wavefront control using, for example, a liquid crystal matrix. Here, the transmission type spatial phase modulator 753 can control the phase of 256 × 256 pixels in accordance with an instruction from the controller 700. Since the other configuration is the same as that of the first embodiment, the difference will be mainly described below.

  When observing a depth position of, for example, 100 μm or more in the sample 714, spherical aberration occurs according to the refractive index of the sample 714, and the focal position is separated according to the aperture radius position of the objective lens 713 as shown in FIG. To do. The aberrations corrected by the first to third embodiments are generally limited to spherical aberrations up to the fourth order. Therefore, even if the movement amount dz1 of the aberration correction lens 752 and the movement amount dz2 of the objective lens are controlled so that the aberration of the light spot is minimized, higher-order aberrations exceeding the fourth order remain.

  This remaining high-order aberration can be calculated in advance. Therefore, the amount of aberration can be further reduced by providing a phase distribution in which the spatial phase modulator 753 cancels the residual aberration. Here, for the sake of simplicity of explanation, an example in which a transmissive spatial phase modulator 753 is used has been described, but a reflective spatial phase modulator 753 can also be easily used.

  FIG. 22 shows a calculation result indicating the phase distribution given by the spatial phase modulator 753. The sample 714 has a refractive index of 1.38 and a measurement depth of 100 μm. The horizontal axis is a normalized radius where the effective radius of the objective lens 713 is 1, and the vertical axis indicates the phase of the wavefront. For comparison, the residual aberration in the first embodiment is also shown. As shown in FIG. 22, the remaining wavefront aberration in the first embodiment is a higher-order aberration having a radius of the fifth or higher. It can be seen that the wavefront synthesized by the spatial phase modulator 753 performing the reverse correction of the residual aberration is almost free of aberration. The residual aberration is due to the limitations on the phase control resolution (here, 8 bits / 1λ) of the spatial phase modulator 753 and the pixel size (here 256 × 256).

  FIG. 23 is a calculation result showing the relationship between the measurement depth inside the sample 714 and the cube of the spot light intensity. Since the intensity of the CARS light is proportional to the cube of the spot light intensity, the vertical axis in FIG. 23 is proportional to the CARS light intensity. Here, the calculation result when the refractive index n of the sample 714 is 1.40 is shown. As shown in FIG. 23, high intensity CARS light can be obtained even at a depth of 1 mm or more by aberration correction with the addition of the spatial phase modulator 753.

  The wavefront provided by the spatial phase modulator 753 can be uniquely determined if the refractive index of the sample 714 and the position control amounts of the aberration correction lens 752 and the objective lens 713 are determined. Therefore, even in the aberration correction optical system equipped with the spatial phase modulator 753, if the wavefront to be applied is set to zero phase, the processing for the new sample having the refractive index n described in the third embodiment can be performed as it is. That is, the wavefront provided by the spatial phase modulator 753 can be uniquely determined together with dz1 and dz2. Aberration correction can be performed according to the flowchart of FIG.

<Embodiment 5>
In Embodiment 5 of the present invention, spherical aberration correction using reflected CARS light will be described. Reflective CARS is a method in which a detector is installed on the incident light side with reference to a sample. In the fifth embodiment, the intensity of the reflected CARS light is detected by a detector, and the CARS spectrum is detected by a transmission spectroscope.

  FIG. 24 is a configuration diagram of an optical measurement apparatus 1000 according to the fifth embodiment. The dichroic mirror 2401 transmits incident light (pump light and Stokes light) and detects CARS light. For example, when the wavelength of the pump light is 1064 nm, a dichroic mirror 2401 may be selected that reflects 1000 nm or more and transmits more wavelengths. Of the CARS light generated from the sample 714, the reflected component is reflected by the dichroic mirror 2401, the residual component of the incident light is removed by the notch filter 2402, and then the reflected CARS light is detected by the detector 2403. As the detector 2403, it is convenient to use a PD (photodiode) or an APD (avalanche photodiode), but a photomultiplier tube may be used when the sensitivity is insufficient. If the wavelength selectivity of the dichroic mirror 2401 is sufficiently good, the notch filter 2402 may be omitted. Since other configurations are the same as those of the first embodiment, description thereof is omitted.

  FIG. 25 is a flowchart for explaining the operation of the optical measurement apparatus 1000 according to the fifth embodiment. Steps S011, S012, and S015 are the same as those in FIG. In step S2501, the controller 700 controls the aberration correction mechanism 750 so that the amount of reflected CARS light detected by the detector 2403 is maximized. In step S2502, the controller 700 corrects the position of the objective lens 713 so that the transmitted CARS light amount detected by the spectroscope 719 is maximized. Thereby, the focal position of the objective lens 713 is adjusted to the focal position of the detection objective lens 715. That is, the focal position moved by the spherical aberration correction is corrected to the original position.

  In the reflection CARS, the condensing objective lens also operates as a detection objective lens. Therefore, the CARS light amount detected by reflection depends only on the condensing state such as the amount of spherical aberration and does not depend directly on the condensing position inside the sample 714. . Therefore, aberration correction can be performed without holding the movement amount of the aberration correction lens 752 corresponding to the measurement depth described in FIG. Further, as described above, the position of the condenser objective lens may be adjusted so that the signal amount of the transmission CARS is maximized. Therefore, it is not necessary to maintain the position of the objective lens 713 with respect to the measurement depth described with reference to FIG. Therefore, the apparatus configuration is simple. In the case of simply detecting the amount of reflected light of incident light, the detection amount is constant regardless of the amount of spherical aberration. However, in the present invention, since the CARS light proportional to the cube of the incident light intensity is detected, the spherical aberration is reduced. Correction can be made with high sensitivity.

<Embodiment 6>
In the sixth embodiment of the present invention, compensation for chromatic aberration occurring in the sample 714 will be described. Since the CARS process is one of nonlinear optical effects, it is important that the pump light and the Stokes light are collected at the same position in the sample 714. In the case of multicolor CARS, the Stokes light has a wide band, so chromatic aberration is generated by the objective lens, and the CARS light spectrum changes depending on the spatial multiplexing state of the pump light and Stokes light in the sample 714.

  FIG. 26 is a configuration diagram of the optical measurement apparatus 1000 according to the sixth embodiment. In the sixth embodiment, the combined state of the pump light and the Stokes light in the sample 714 is adjusted by adjusting the divergent convergence state of the Stokes light. The stage 2601 controls the divergent convergence state of the Stokes light by adjusting the position of the collimating lens 706 in the optical axis direction (z direction) in accordance with an instruction from the controller 700. In the sample 714, the converging position changes due to the divergent convergence state, whereby the combined state changes, and the shape of the CARS light spectrum changes. Since the other apparatus configuration is the same as that of the first embodiment, description thereof is omitted.

  FIG. 27 shows a modification of the sixth embodiment. FIG. 26 shows an example of adjusting the divergence / convergence state of Stokes light. However, as shown in FIG. 27, a fiber optical system is also used in the pump light path, and the collimator lens 2603 and the stage 2604 are used at the output end of the optical fiber 2602. The divergence convergence state may be controlled. Furthermore, the divergent convergence state may be controlled for both pump light and Stokes light by combining FIG. 26 and FIG.

  FIG. 28 is a flowchart for explaining the operation of the optical measurement apparatus 1000 according to the sixth embodiment. 20 is the same as FIG. 20 except that step S2701 is added between steps S014 and S015. In step S2701, the controller 700 detects the CARS spectrum by the spectroscope 719, and adjusts the position of the collimating lens 706 (or the collimating lens 2603 in addition to this) so that a desired wavelength band is enhanced. The adjustment completion threshold value may be determined by the absolute value of the peak intensity of the spectrum, or may be determined from the ratio with other spectrum peaks.

FIG. 29 shows the result of comparison of CARS spectra of fat cells before and after chromatic aberration correction described in the sixth embodiment. In the CARS spectrum, in addition to a strong spectral peak corresponding to CH stretching near 2900 cm ^−1, there are a plurality of peaks such as amide III, CH deflection, amide I, etc. in the region of about 800 to 1800 cm ⁻¹ . . The latter region is called a fingerprint region and is useful for analyzing the cell state because it corresponds to a plurality of compositions of the living body, but the problem is that the signal intensity is low.

  In the spectrum 2801 before correction, the signal corresponding to the CH expansion / contraction is obtained with high intensity, but the signal in the fingerprint region is hardly obtained. On the other hand, in the corrected spectrum 2802, the spectrum peak in the fingerprint region is emphasized. As described above, the optical measurement apparatus 1000 according to the sixth embodiment can emphasize the CARS signal intensity in the desired frequency band.

  According to the sixth embodiment, the frequency band to be observed is notified to the controller 700 via an appropriate interface 1110 (for example, an operating device, a data input interface, etc.), and the controller 700 indicates the CARS signal strength of the frequency band. Each optical element (the positions of the collimating lenses 706 and 2603) can be controlled so as to be emphasized.

<Embodiment 7>
FIG. 30 is a schematic diagram showing an aberration correction mechanism 750 according to Embodiment 7 of the present invention. The sample 714 has a relatively thin thickness of about 100 μm or less. In the seventh embodiment, an objective lens 713 and a detection lens 781 are arranged so as to sandwich the sample 714. CARS light (signal light) generated in the vicinity of the focal point of the objective lens 713 is converted into a parallel light beam by a detection lens 781, and a spectrum is measured by a spectroscope (not shown). Other configurations are the same as those of the first embodiment.

  FIG. 31 is a schematic diagram illustrating another configuration example of the aberration correction mechanism 750 according to the seventh embodiment. The sample 714 has a relatively thick thickness of about 100 μm or more. When the thickness of the sample 714 is t and the measurement depth is z, the light beam emitted from the objective lens 713 is affected by spherical aberration due to the thickness z of the sample 714. The CARS light (signal light) acquired by the detection lens 781 is affected by spherical aberration due to the thickness (tz) of the sample 714. Therefore, a shift of δz occurs between the focal position of the objective lens 713 and the focal position of the detection lens 781.

  The controller 700 once again sets the measurement depth z as the focal position of the detection lens 781, and controls the position of the objective lens 713 by the actuator 762 so that the focal position of the detection lens 781 and the focal position of the objective lens 713 coincide. Thereby, a large CARS signal can be acquired with high spectral resolution without depending on the measurement depth z.

  FIG. 32 shows the result of calculating the amount of movement for recorrecting the position of the objective lens 713. It is assumed that the refractive index of the sample 714 is n, and the amount of re-movement of the objective lens 713 when the position of the objective lens 713 is recorrected to match the focal position of the objective lens 713 and the focal position of the detection lens 781 is dz2 '. For simplification of description, the same immersion lens (numerical aperture 1.2) as that of the objective lens 713 is used as the detection lens 781. The thickness t of the sample 714 is 500 μm.

  As shown in FIG. 32, it can be seen that the re-movement amount dz2 'of the objective lens 713 is determined by the refractive index of the sample 714, and the dependence on the measurement depth z is relatively small. By controlling the position of the aberration correction lens 752 by dz1 and by controlling the position of the objective lens 713 by dz2 + dz2 ', it is possible to correct the influence of aberration caused by the thickness of the sample 714.

  Since dz2 ′ is determined according to the refractive index and thickness of the sample 714, dz2 ′ is described for each combination of the refractive index and thickness of the sample 714 in the data table stored in the storage unit 1100. Dz2 ′ can be obtained according to the description.

700 Controller 701 Short pulse laser light source 702 Half-wave plate 703 Polarizing beam splitter 704 Condensing lens 705 Photonic crystal fiber 706 Collimate lens 707 Long pass filter 708 Dichroic mirror 709 Mirror 710 Lens 711 Lens 712 Mirror 713 Objective lens 714 Sample 715 Objective Lens 716 Collimate lens 717 Notch filter 718 Short pass filter 719 Spectroscope 720 Condensing lens 721 Optical fiber 722 Collimate lens 723 Spatial phase modulator 724 Long pass filter 750 Aberration correction mechanism 751 Aberration correction lens 752 Aberration correction lens 753 Spatial phase modulator 761 Actuator 762 Actuator 763 Actuator 781 Detection lens 2401 Dyke Loic mirror 2402 Notch filter 2403 Detector 2602 Optical fiber 2603 Collimate lens 2604 Stage

Claims (7)

A light source that emits laser light,
A branching portion for branching the laser light emitted from the light source into first light and second light;
An optical fiber that propagates the first light;
A multiplexing unit that generates combined light by combining the second light and the first light propagated by the optical fiber;
An objective lens for irradiating the sample with the combined light;
A detector for detecting signal light generated by irradiating the sample with the combined light;
A spherical aberration correction unit that corrects spherical aberration generated according to the thickness of the sample using an aberration correction lens disposed on an optical path between the light source and the objective lens,
The positional relationship between the aberration correction lens and the objective lens on the optical path is such that the aberration correction lens can correct the spherical aberration while suppressing the focal position of the combined light in the sample within a predetermined range. A storage unit for storing the described data;
With
The spherical aberration correction unit corrects the spherical aberration while suppressing the focal position within the predetermined range by controlling the position of the aberration correction lens according to the positional relationship described by the data. A characteristic optical measuring device. The data describes the positional relationship for the first refractive index of the first sample,
The spherical aberration correction unit obtains a first difference between the second refractive index of the second sample and the refractive index of the peripheral medium of the objective lens,
The spherical aberration correction unit obtains a second difference between the first refractive index and the refractive index of the peripheral medium,
The spherical aberration corrector corrects the aberration according to a second positional relationship obtained by multiplying the positional relationship of the first sample with respect to the first refractive index by a ratio of the first difference to the second difference. The optical measurement apparatus according to claim 1, wherein the spherical aberration of the second sample is corrected by controlling a position of a correction lens. The optical measurement device further includes a second detector that measures light reflected from the sample,
The data describes the positional relationship for the first refractive index of the first sample and describes the positional relationship for each detected depth of the sample,
The spherical aberration correction unit specifies a positional relationship between the objective lens and the aberration correction lens when the second detector detects a maximum light intensity,
The spherical aberration correction unit acquires the positional relationship corresponding to the detection depth of the sample when the second detector detects the maximum light intensity from the data,
The spherical aberration correction unit obtains the second refractive index of the second sample using the positional relationship acquired from the data, the first refractive index, and the refractive index of the peripheral medium of the objective lens. The optical measurement device according to claim 1. The optical measurement device further includes a detection lens that receives the signal light,
The sample is disposed at a position sandwiched between the light source and the detection lens,
The detector detects the signal light received by the detection lens;
The data further describes a recorrection amount for recorrecting the position of the objective lens for each combination of the thickness of the sample and the refractive index of the sample.
The optical measurement apparatus according to claim 1, wherein the spherical aberration correction unit controls the position of the objective lens after adding the recorrection amount to the positional relationship. The optical measurement device according to claim 1, further comprising a spatial phase modulator that corrects the remaining aberration after the spherical aberration correction unit corrects the spherical aberration. The optical measuring device further includes
An interface that specifies the light wavelength to be observed,
An optical element that adjusts the combined state of the combined light so that the intensity of the signal light at the specified optical wavelength is maximized;
The optical measurement device according to claim 1, further comprising: The optical measurement apparatus according to claim 1, wherein the detector is configured as a spectrometer that detects a wavelength spectrum of light generated from the sample.

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