JP6850684B2 - Optical measuring device - Google Patents

Description

The present invention relates to an optical measuring device that measures a sample by irradiating it with light.

Optical microscopes have evolved as essential devices in the natural sciences, engineering, and industrial fields. In particular, in recent years, an optical microscope may be 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 the current cell analysis, it is common to stain cells with a reagent and then observe them with a microscope or the like. However, this method has an effect on cells by staining, so it is difficult to continuously analyze the same cells or to directly use the examined cells for medical purposes.

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

The following Patent Document 1 describes a configuration example for detecting only a specific spectral component of CARS light. Patent Document 2 below describes a multicolor CARS microscope that spectroscopically detects CARS light generated by using a wideband light source. Since the multicolor CARS microscope can estimate the Raman spectrum from the spectral spectrum of CARS light, the amount of information that can be acquired is large, and the measurement target can be analyzed in more detail.

The following Patent Document 3 discloses a method of identifying and correcting a spherical error when an image of a sample is taken with a microscope. In the same document, the control unit corrects the spherical error by electrically adjusting the correction lens element. Furthermore, the autofocus control device is used to eliminate the defocus situation that occurs during microscopic examination.

U.S. Pat. No. 6,108,081 Japanese Unexamined Patent Publication No. 2009-222531 Japanese Unexamined Patent Publication No. 2013-0888009

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

In CARS, when cells are moved along the optical axis direction for imaging, the sample thickness between the focusing objective lens and the focusing position changes due to the movement, which causes spherical aberration. In order to secure the signal strength, dynamic spherical aberration correction corresponding to the position of the focusing objective lens is effective. However, on the other hand, when spherical aberration correction is performed, the focusing position changes due to the spherical aberration correction. Therefore, the CARS light generation point deviates from the focal position of the detection objective lens, and the signal intensity decreases.

The present invention has been made in view of the above problems, and an object of the present invention is to provide an optical measuring device capable of correcting a focus shift due to a correction of spherical aberration and ensuring a signal intensity. To do.

The optical measuring device according to the present invention provides data describing the positional relationship between the aberration correction lens and the objective lens, which can correct spherical aberration while keeping the focal position of the combined wave light in the sample within a predetermined range. It is stored in advance, and the spherical aberration is corrected according to the data.

According to the optical measuring apparatus according to the present invention, even when the inside of the sample is measured by CARS, the decrease in signal intensity is suppressed 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.} This is a process example when a wideband light source is used. 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 apparatus 1000 which concerns on Embodiment 1. FIG. It is a schematic diagram which shows the detail of the aberration correction by the aberration correction mechanism 750. This 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. This is a calculation result showing the relationship between the measurement depth inside the sample 714 and the cube of the spot light intensity. The calculation result of the movement amount dz1 of the aberration correction lens 752 used when carrying out the calculation shown in FIG. 8 is shown. The calculation result of the movement amount dz2 of the objective lens 713 used when carrying out the calculation shown in FIG. 8 is shown. The change in the focal position in the calculation result shown in FIG. 8 is shown. The result of calculating dz1 (z, n0) according to the formula 3 is shown. The result of calculating dz2 (z, n0) according to the formula 4 is shown. The calculation result of the spot light intensity when the aberration correction lens 752 and the objective lens 713 are moved at the same time according to the equations 1 and 2 to correct the aberration is shown. The change in the light spot position when the aberration correction lens 752 and the objective lens 713 are moved at the same time according to the equations 1 and 2 to correct the aberration is shown. It is a schematic diagram which shows the detail of the aberration correction by the aberration correction mechanism 750 which concerns on Embodiment 3. It shows how to make the measurement depth in the sample a desired value. The state of controlling dz1 is shown. The relationship between the wave surface aberration and the cube value of the spot light intensity is shown. It is a flowchart which shows the control procedure by the controller 700 in Embodiments 1-3. It is a schematic diagram of the aberration correction mechanism 750 according to the fourth embodiment. The calculation result which shows the phase distribution given by the spatial phase modulator 753 is shown. This is a calculation result showing 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 apparatus 1000 which concerns on Embodiment 5. It is a flowchart explaining the operation of the optical measuring apparatus 1000 which concerns on Embodiment 5. It is a block diagram of the optical measuring apparatus 1000 which concerns on Embodiment 6. This is a modification of the sixth embodiment. It is a flowchart explaining the operation of the optical measuring apparatus 1000 which concerns on Embodiment 6. It is a result of comparing the CARS spectrum of the adipocyte before and after the chromatic aberration correction described in the sixth embodiment. It is a schematic diagram which shows the aberration correction mechanism 750 which concerns on Embodiment 7. It is a schematic diagram which shows another structural example of the aberration correction mechanism 750 which concerns on Embodiment 7. The result of calculating the movement amount for re-correcting the position of the objective lens 713 is shown.

<About the basic principle of CARS microscope>
In the following, in order to promote the understanding of the present invention, first, the basic principle of the CARS microscope and the problems associated with correcting spherical aberration will be outlined. Hereinafter, embodiments of the present invention will be described.

CARS is light emission due to third-order polarization. In order to generate CARS light, pump light, Stokes light, and probe light are required. Generally, pump light is substituted for probe light in order to reduce the number of light sources. In this case, the induced tertiary polarization is represented by the following formula A. χ _r ⁽³⁾ (ω _AS ) is the resonance term of the molecular vibration of the third-order electric susceptibility, and χ _nr ^{(3), which} has no frequency dependence, is the non-resonance term. It 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 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 with reference to the energy level diagram of the molecule shown in FIG. FIG. 1 shows the process of the resonance term in Equation 1. Reference numeral 2001 represents the ground state of the molecule, and reference numeral 2002 represents the vibrationally excited state. Simultaneously irradiate pump light with frequency ω _{P and Stokes light with frequency ω S.} At this time, the molecule is excited to any of the oscillating excited states 2002 via the intermediate state 2003. When the excited molecule is irradiated with the probe light _{of the frequency ω P} , the molecule returns to the ground state 2001 while generating the CARS light of the _{frequency ω AS through the intermediate state 2004.} The frequency of 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) in the third-order polarization.} 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 the probe light of frequency ω _P , non-resonant CARS light of _{frequency ω AS is generated through the intermediate state 2004.}

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

FIG. 4 is a block diagram of a conventional multicolor CARS microscope. The output from the short pulse laser light source 2301 is split into two by the beam splitter 2302. One of the branched lights is introduced into an optical fiber such as a photonic crystal fiber 2303, and a wide band light (called supercontinuum light) is generated inside the optical fiber. When the super-continue 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), which is used as Stokes light. The other excitation light and Stokes light are combined by the dichroic mirror 2305. The combined wave light is focused and irradiated on the sample 2306 by the focusing objective lens 2309. As a result, 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 spectroscope 2307 detects the CARS light and acquires a wavelength spectrum.

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

As described above, when obtaining the CARS spectrum image, it is common to move the position of the sample 2306 instead of scanning the incident light. The reason for this is that it is easy to match the focal position of the condensing objective lens 2309 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 focusing objective lens 2309 in order to secure the power density. In this case, spherical aberration occurs in which the focusing position of the light passing near the center of the lens and the focusing position of the light passing through the peripheral portion of the lens deviate from each other. Therefore, the spherical aberration is corrected by an aberration correction lens or the like. However, by correcting the spherical aberration, the focal position of the condensing objective lens 2309 deviates 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 view showing a configuration example of the optical measuring device 1000 according to the first embodiment of the present invention. The optical measuring device 1000 includes a beam expander type aberration correction mechanism 750. The aberration correction mechanism 750 operates according to an instruction from the controller 700. The controller 700 includes an input interface (for example, interface 1110 described later) for receiving measurement instructions from the user and an output interface for displaying measurement results, while controlling the overall operation of the optical measurement device 1000.

The short pulse laser light source 701 emits a short pulse laser light according to 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 non-linear optical effect. The wavelength may be appropriately selected based on the absorption band to be measured, the wavelength specifications of the optical components to be used, and the like. For example, wavelengths such as 800 nm and 1064 nm can be used.

The laser beam is incident on the 1/2 wave plate 702 and the polarization beam splitter 703. The 1/2 wave plate 702 changes the polarization direction of the laser beam according to the instruction from the controller 700. The polarization beam splitter 703 splits the laser beam into a transmission component and a reflection component at a power branching ratio based on the polarization direction.

The laser light transmitted through the polarizing beam splitter 703 is focused 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-shaped hollow clad is formed around the core, and the incident light is strongly confined in the core. By injecting a short pulse laser beam onto the photonic crystal fiber 705, a nonlinear optical phenomenon such as self-phase modulation or four-wave mixing is induced, whereby supercontinue light having a wide spectrum is generated.

The generated super-continuum light becomes parallel light by the collimated lens 706, and after the short wavelength component is cut by the long-pass filter 707, it 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 corrects the wave surface of the transmitted light according to the type of the sample 714 and the measurement depth. The corrected light is incident on the objective lens 713 as Stokes light.

The laser beam reflected by the polarizing beam splitter 703 is reflected by the mirror 709, the mirror 712, and the dichroic mirror 708, and is similarly corrected by the aberration correction mechanism 750 and then incident on the objective lens 713 as pump light. To do.

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

In sample 714, the above-mentioned CARS process is induced to generate CARS light having a wavelength corresponding to the molecular species of sample 714. The collimating lens 716 converts CARS light into parallel light. The notch filter 717 and the short pass filter 718 cut the transmission components of the pump light and the 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. The aberration correction mechanism 750 uses a drive mechanism (not shown) to move one of the aberration correction lenses in the optical axis direction according to the type (refractive index) of the sample 714 and the measurement depth according to the instruction from the controller 700. 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 change, 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 aberration correction lenses 751 and 752 as the aberration correction mechanism 750 is shown. These lenses constitute a beam expander optical system that shares 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 the 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 the instruction 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 to correct the spherical aberration while keeping the focal position inside the sample 714 constant. Can be done.

Hereinafter, the effect of the present invention will be verified by calculating the wave surface aberration and the spot light intensity using the ray tracing method. Unless otherwise specified, the optical conditions are as follows: the wavelength of the pump light is 1064 nm, the objective lens 713 has a numerical aperture of 1.2 for the water immersion type, 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 general cover glass is 1.52. According to publicly known literature (Biomedical Photonics Handbook 2nd Edition, volume1, CRC Press, Tuan Vo-Dinh.), The refractive index of human tissues is the brain, heart, except for special tissues such as eyes, keratin, bones, and teeth. It is 1.36 to 1.38 in major tissues such as kidney, spleen, stomach and muscle, and 1.39 to 1.40 in bladder wall and arteries. Since cells such as iPS cells and ES cells are cultured in a culture medium, the refractive index is close to that of water, and it is considered that the refractive index is about 1.333 to 1.35 depending on the cell density and the state of the culture medium. Can be done.

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. The result. The refractive index of 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 wave surface aberration can be minimized without changing the focal position. When the refractive index of sample 714 changes, the relationship shown in FIG. 7 also changes, but as will be described later, a 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 CARS light is proportional to the cube of the spot light intensity, the vertical axis of FIG. 8 is proportional to the intensity of CARS light.

FIG. 8A shows the result when the aberration is not corrected. It can be seen that when the refractive index of the sample 714 is 1.36 to 1.38 (corresponding to the main tissue of the living body), the intensity of CARS light is attenuated to 1/2 or less at a depth of about 50 μm.

FIG. 8B shows a calculation result when the aberration correction lens 752 and the objective lens 713 are moved at the same time to correct the aberration according to the relationship shown in FIG. It can be seen that in the range of the refractive index of sample 714 in the range of 1.34 to 1.39, the intensity of CARS light can be maintained at 1/2 or more over a depth of 500 μm or more. When the cell concentration is low as in 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 1/2 or more is up to a depth of about 400 μm. It can be seen that the refractive index of 1.525 corresponds to the case of a general cover glass, and that aberration correction can be performed only up to a depth of about 100 μm.

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

FIG. 10 shows the 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 sample 714 increases.

FIG. 11 shows the 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 index of refraction of sample 714 ranges from 1.333 to 1.40.

<Embodiment 1: Summary>
The optical measuring device 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. As a result, high-intensity CARS light can be acquired at a measurement depth of 100 μm or more.

Some objective lenses used in conventional microscopes are provided with 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 will change due to aberration correction. In addition, the refractive index of the standardized inorganic cover glass is constant, and it is difficult to measure while maintaining a high CARS light intensity inside the biological tissue whose refractive index changes depending on the type and culture state of cells and tissues. Is. According to the first embodiment, it is advantageous in that even such a sample can maintain a large CARS light intensity.

<Embodiment 2>
Unlike the case of the cover glass, which is an inorganic substance, in the biological sample, the refractive index varies with a continuous numerical value depending on the sample individual or the conditions such as the number of culture days. If the refractive index of the same sample changes, it can be regarded as a different sample. In the second embodiment of the present invention, a method of correcting the aberration according to the change in the refractive index of such a biological sample will be described. The configuration of the optical measuring 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 (refractive index of a certain sample). Assuming that the refractive index of the peripheral medium (assuming water here) of the objective lens 713 is n _WATER , these functions dz1 (z, n) and dz2 (z, n) for a new sample having a 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 approximation by Equation 1 and Equation 2 is valid, the following Equations 3 and 4 obtained by modifying Equation 1 and Equation 2 are used as dz1 (z, n0). Actually calculate dz2 (z, n0).

FIG. 12 shows the result of calculating dz1 (z, n0) according to the equation 3. In FIG. 12, n0 = 1.38 was set, and the calculation result of dz1 (z, n) shown in FIG. 9 was 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). That is, it can be seen from the approximation of Equation 1 that aberration correction is possible according to an arbitrary refractive index of the biological sample. The refractive index n = 1.525 corresponds to a general cover glass, and the difference in characteristics is large, so that it cannot be approximated. In other words, the conventional technique used for correcting the cover glass thickness corresponds to the curve of the refractive index n = 1.525 in FIG. 12, which is different from the aberration correction method in the present invention. It can be said that.

FIG. 13 shows the result of calculating dz2 (z, n0) according to the equation 4. In FIG. 13, n0 = 1.38 was set, and the calculation result of dz2 (z, n) shown in FIG. 10 was 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 difference in characteristics is large as in FIG.

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

FIG. 14 shows the calculation result of the spot light intensity when the aberration correction lens 752 and the objective lens 713 are moved at the same time according to the equations 1 and 2 to correct the aberration. The horizontal axis represents the measurement 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 value 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, in the case of a refractive index of 1.525, which corresponds to a general cover glass, the attenuation of CARS light intensity is large.

FIG. 15 shows a change in the position of the light spot when the aberration correction lens 752 and the objective lens 713 are simultaneously moved according to the equations 1 and 2 to correct the aberration. In the range of refractive index 1.34 to 1.40, the change in 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, in the case of a refractive index of 1.525, which corresponds to a general cover glass, the deviation of the focal position is large.

<Embodiment 3>
FIG. 16 is a schematic view showing 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 is the boundary surface of the sample 714. The controller 700 controls the aberration correction lens 752 and the objective lens 713 to the origin position where the aberration correction amount becomes zero by the actuators 761 and 762. Since other configurations are the same as those in the first embodiment, the differences will be mainly described below.

When the pump light is applied to 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. At this time, the position of the actuator 763 in the optical axis direction is set to dz3 = 0.

FIG. 17 shows how the measurement depth in the sample is set to 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 aperture position of the objective lens 713 due to the aberration corresponding 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. As a result, 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 wave surface aberration and the cube value 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 collected by the lens 771, and is received by the light detector 772. FIG. 19A shows the relationship between the position dz1 of the aberration correction lens 752 and the wave surface 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 cube aberration of the wave surface aberration and the spot light intensity under each condition of z = 100, 200, 300, 400, and 500 μm. The refractive index of sample 714 is n0 = 1.38. For example, when z = 200 μm, when dz1 = 665 μm, the wave surface aberration is the minimum, the cube value of the spot light intensity is the maximum, and the amount of CARS light generated is the maximum. Since dz1 (z, n0) matches the focal point of the objective lens 713 with the measurement depth while correcting the aberration, the amount of movement of the aberration correction lens 752 at this time is dz1 (z, n0). become.

Similarly, for another sample having a refractive index of n, the amount of movement of the aberration correction lens 751 is dz1 (z, n) when the CARS light intensity detected by the photodetector 772 becomes maximum. The refractive index n can be obtained by the following formula 5 which is a modification of the formula 1. In Equation 5, the right side is the known value dz1 (z, n0) and dz1 (z, n) when the light intensity is maximum, and the left side is a known value other than 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 α is also contained in the right side of the equation 1 and the right side of the equation 2, by using the α obtained as the actual measurement result according to the third embodiment, the measurement depth inside the sample 714 is changed to dz1. There is no need to adjust dz2. That is, dz1 and dz2 can be calculated according to Equations 1, 2, and α at the measurement depth inside any sample 714.

FIG. 20 is a flowchart showing 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 amount of CARS light 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 measurement depth inside the designated 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 the sake of brevity, the sample 714 has a uniform refractive index in the depth direction. On the other hand, many samples such as actual biological tissues and cell sheets have a layered structure within the measurement depth range. In this case, the conditions for maximizing the amount of CARS light described in FIGS. 16 to 18 were implemented at each measurement depth to correspond to the average refractive index of the medium until the pump light and the Stokes light reach the focal point. Find the control amount. For any measurement depth inside the sample 714, the control amount can be determined by interpolating them.

The third embodiment shows a method of correcting aberrations using photodetectors 772 and 774. In the third embodiment, it is also effective to arrange pinholes 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 the first to third embodiments, the representative refractive index is n0 = 1.38, but as shown in FIGS. 12 and 13, the refraction of the sample having a high evaluation frequency is within the range where the approximation between the equations 1 and 2 is established. Needless to say, the control accuracy is improved by selecting the rate as the representative refractive index.

In the third embodiment, it is possible to detect the sample 714 boundary and obtain the condition for maximizing the amount of CARS light without using the photodetectors 772 and 774. In the optical measuring device 1000 shown in FIG. 1, a microscope optical system is added to the optical system of the detection objective lens 715, and a general method such as a phase difference method is used so that the contrast of the microscope image at the boundary of the sample 714 is maximized. The position of the actuator 763 can be adjusted to determine the boundary position of the sample 714. Thereby, the function of the photodetector 774 can be substituted. After moving the sample 714 to a predetermined measurement depth, the photodetector 772 is controlled by controlling the position of the aberration correction lens 752 and the position of the objective lens 713 so that the amount of light received by the spectroscope 719 is maximized. The function 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, and the present embodiment 3 It is also possible to automatically control dz1 and dz2 using the data table corresponding to the refractive index n obtained according to the above.

<Embodiment 4>
FIG. 21 is a schematic view of the aberration correction mechanism 750 according to the fourth embodiment 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 arbitrarily controlling the wave surface by, for example, a liquid crystal matrix. Here, the transmissive spatial phase modulator 753 can perform phase control of 256 x 256 pixels according to an instruction from the controller 700. Since other configurations are the same as those in the first embodiment, the differences 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 as to minimize the aberration of the light spot, higher-order aberrations exceeding the fourth order remain.

This residual 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 simplification of the description, an example of using the transmission type spatial phase modulator 753 is shown, but it is also easy to use the reflection type spatial phase modulator 753.

FIG. 22 shows a calculation result showing the phase distribution provided by the spatial phase modulator 753. The refractive index of sample 714 is 1.38, and the measurement depth is 100 μm. The horizontal axis is the normalized radius with the effective radius of the objective lens 713 as 1, and the vertical axis indicates the phase of the wave surface. For comparison, the residual aberrations in the first embodiment are also shown. As shown in FIG. 22, the residual wave surface aberration in the first embodiment is a high-order aberration of the fifth order or higher of the radius. It can be seen that the wave surface synthesized by the spatial phase modulator 753 performing the inverse correction of the residual aberration is almost free of aberration. The residual aberration is due to the limitation of the phase control resolution (here, 8 bits / 1λ) and the pixel size (here, 256x256) of the spatial phase modulator 753.

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 CARS light is proportional to the cube of the spot light intensity, the vertical axis of FIG. 23 is proportional to the intensity of CARS light. Here, the calculation result when the refractive index n = 1.40 of the sample 714 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 wave surface imparted by the spatial phase modulator 753 can be uniquely obtained 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 applied wave surface is set to zero phase, the processing for the new sample having the refractive index n described in the third embodiment can be carried out as it is. That is, together with dz1 and dz2, the wave surface imparted by the spatial phase modulator 753 can be uniquely determined. 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. Reflection 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 type spectroscope.

FIG. 24 is a configuration diagram of the optical measuring device 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 that reflects 1000 nm or more and transmits a wavelength higher than that may be selected. 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 if the sensitivity is insufficient, a photomultiplier tube may be used. 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 in the first embodiment, the description thereof will be omitted.

FIG. 25 is a flowchart illustrating the operation of the optical measuring device 1000 according to the fifth embodiment. Steps S011, S012, and S015 are the same as 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 amount of transmitted CARS light detected by the spectroscope 719 is maximized. As a result, 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, since the focusing objective lens also operates as a detection objective lens, the amount of CARS light detected by reflection depends only on the focusing state such as the amount of spherical aberration, and does not directly depend on the focusing position inside the sample 714. .. Therefore, the aberration can be corrected even if the movement amount of the aberration correction lens 752 corresponding to the measurement depth described with reference to FIG. 9 is not maintained. Further, as described above, the position of the condensing objective lens may be adjusted so that the signal amount of the transmitted CARS is maximized, so that 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 device configuration becomes simple. When simply detecting the reflected light amount of the incident light, the detection amount is constant regardless of the amount of spherical aberration. However, in the present invention, CARS light proportional to the cube of the incident light intensity is detected, so that spherical aberration can be prevented. It can be corrected with good sensitivity.

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

FIG. 26 is a configuration diagram of the optical measuring device 1000 according to the sixth embodiment. In the sixth embodiment, the divergence and convergence state of the Stokes light is adjusted to adjust the combined state of the pump light and the Stokes light inside the sample 714. The stage 2601 controls the divergence and convergence state of the Stokes light by adjusting the position of the collimating lens 706 in the optical axis direction (z direction) according to the instruction from the controller 700. Inside the sample 714, the converging state changes due to the change in the focusing position due to this divergence and convergence state, and the shape of the CARS optical spectrum changes. Since other device configurations are the same as those in the first embodiment, the description thereof will be omitted.

FIG. 27 is a modified example of the sixth embodiment. FIG. 26 shows an example of adjusting the divergence and convergence state of Stokes light. As shown in FIG. 27, a fiber optical system is also adopted for the pump optical path, and a collimating lens 2603 and a stage 2604 are used at the exit end of the optical fiber 2602. The divergence convergence state may be controlled. Further, the divergence and convergence state of both the pump light and the Stokes light may be controlled by combining FIGS. 26 and 27.

FIG. 28 is a flowchart illustrating the operation of the optical measuring device 1000 according to the sixth embodiment. This is the same as in 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 additionally the collimating lens 2603) so that the desired wavelength band is emphasized. The adjustment completion threshold value may be determined by the absolute value of the peak intensity of the spectrum, or may be determined by the ratio with other spectrum peaks.

FIG. 29 shows the results of comparing the CARS spectra of adipocytes before and after the chromatic aberration correction described in the sixth embodiment. The CARS spectrum, in addition to the strong spectral peaks corresponding to CH stretching of 2900 cm ^-1 vicinity amide III in the region from about 800～1800Cm ^-1, CH bending, there are several peaks of an amide I .. The latter region is called the fingerprint region and is useful in the analysis of cell state because it corresponds to a plurality of compositions of a living body, but the problem is that the signal strength is low.

In the spectrum 2801 before correction, a signal corresponding to CH expansion and contraction is obtained with high intensity, but a 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 measuring device 1000 according to the sixth embodiment can emphasize the CARS signal intensity in the desired frequency band.

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

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

FIG. 31 is a schematic view showing another configuration example of the aberration correction mechanism 750 according to the seventh embodiment. The thickness of the sample 714 is relatively thick, about 100 μm or more. Assuming that the thickness of the sample 714 is t and the measurement depth is z, the luminous flux emitted from the objective lens 713 is affected by the spherical aberration due to the thickness z of the sample 714. Further, 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 deviation of δz occurs between the focal position of the objective lens 713 and the focal position of the detection lens 781.

The controller 700 again determines 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 match. As a result, a large CARS signal can be acquired with high spectral resolution regardless of the measurement depth z.

FIG. 32 shows the result of calculating the movement amount for re-correcting the position of the objective lens 713. The refractive index of the sample 714 is n, and the re-movement amount of the objective lens 713 when the position of the objective lens 713 is re-corrected in order to match the focal position of the objective lens 713 with the focal position of the detection lens 781 is dz2'. For simplification of the explanation, the same water immersion lens (numerical aperture 1.2) as the objective lens 713 was used as the detection lens 781. The thickness t of the sample 714 is 500 μm.

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

Since dz2'is determined according to the refractive index and thickness of sample 714, dz2'is described for each combination of refractive index and thickness of sample 714 in the data table stored in the storage unit 1100, and the controller 700 is used. According to the description, dz2'can be obtained.

700 Controller 701 Short pulse laser light source 702 1/2 wavelength plate 703 Polarized beam splitter 704 Condensing lens 705 Photonic crystal fiber 706 Collimating lens 707 Long pass filter 708 Dycroic mirror 709 Mirror 710 Lens 711 Lens 712 Mirror 713 Objective lens 714 Sample 715 Objective Lens 716 Collimating lens 717 Notch filter 718 Short pass filter 719 Spectrometer 720 Condensing lens 721 Optical fiber 722 Collimating lens 723 Spatial phase modulator 724 Long pass filter 750 Abrasiveness correction mechanism 751 Abrasiveness correction lens 752 Abrasiveness correction lens 753 Spatial phase modulator 761 Actuator 762 Actuator 763 Actuator 781 Detection lens 2401 Dycroic mirror 2402 Notch filter 2403 Detector 2602 Optical fiber 2603 Collimating lens 2604 Stage

Claims (7)

A light source that emits laser light,
A branching portion that branches the laser light emitted by the light source into the first light and the second light.
An optical fiber that propagates the first light,
A combiner that generates combined light by combining the second light and the first light propagated by the optical fiber.
An objective lens that irradiates a sample with the combined wave light,
A detector that detects signal light generated by irradiating the sample with the combined wave light.
A spherical aberration corrector, which corrects spherical aberration generated according to the thickness of the sample by using an aberration correction lens arranged 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, in which the aberration correction lens can correct the spherical aberration while keeping the focal position of the combined wave light in the sample within a predetermined range. A storage unit that stores the described data,
With
The data describes the positional relationship for each measurement depth position inside the sample.
When the measurement depth position inside the sample changes by controlling the positions of the aberration correction lens and the objective lens according to the positional relationship described by the data. Even if there is, an optical measuring device characterized in that the spherical aberration is corrected while keeping the focal position within the predetermined range. The data describes the positional relationship with respect to the first refractive index of the first sample.
The spherical aberration correction unit obtains the 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 has the aberration according to the second positional relationship obtained by multiplying the ratio of the first difference to the second difference by the positional relationship of the first sample with respect to the first refractive index. The optical measuring device according to claim 1, wherein the spherical aberration of the second sample is corrected by controlling the position of the correction lens. The optical measuring device further includes a second detector that measures the light reflected from the sample.
The data describes the positional relationship with respect to the first refractive index of the first sample, and also describes the positional relationship for each detection depth of the sample.
The spherical aberration correction unit identifies the positional relationship between the objective lens and the aberration correction lens when the second detector detects the 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 is characterized in that the second refractive index of the second sample is obtained by 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 measuring device according to claim 1. The optical measuring device further includes a detection lens that receives the signal light.
The sample is arranged at a position sandwiched between the light source and the detection lens.
The detector detects the signal light received by the detection lens, and receives the signal light.
The data further describes the amount of re-correction for re-correcting the position of the objective lens for each combination of the thickness of the sample and the refractive index of the sample.
The optical measuring device according to claim 1, wherein the spherical aberration correction unit controls the position of the objective lens after adding the re-correction amount to the positional relationship. The optical measuring device according to claim 1, further comprising a spatial phase modulator in which the spherical aberration correcting unit corrects the spherical aberration and then corrects the residual aberration. The optical measuring device further
An interface that specifies the optical 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 light wavelength is maximized.
The optical measuring device according to claim 1, further comprising. The optical measuring device according to claim 1, wherein the detector is configured as a spectroscope that detects a wavelength spectrum of light generated from the sample.

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