WO2012127880A1

WO2012127880A1 - Observation device and observation method

Info

Publication number: WO2012127880A1
Application number: PCT/JP2012/002048
Authority: WO
Inventors: 久美子西村; 中山　繁
Original assignee: 株式会社ニコン
Priority date: 2011-03-24
Filing date: 2012-03-23
Publication date: 2012-09-27
Also published as: JP5610063B2; JPWO2012127880A1

Abstract

An observation device is provided with: a branch means for branching incident light into reference light and measurement light; an illumination optical system that irradiates a subject to be observed with the measurement light along the predetermined direction; an observation optical system that is arranged on a side different from the illumination optical system with respect to the subject to be observed, and receives observation light traveling toward the direction different from the predetermined direction among the measurement light passing through the subject to be observed; a combination optical system that guides the reference light to the observation optical system to combine the reference light and the observation light; and a detection device that receives the reference light and the observation light, which are combined by the combination optical system, to detect the interference intensity of the reference light and the observation light, thereby enabling detection on the basis of light transmitting through the subject to be observed.

Description

Observation apparatus and observation method

The present invention relates to an observation apparatus and an observation method.

There is optical coherence tomography (OCT) as one of the non-destructive tomographic measurement technologies (see Non-Patent Document 1, etc.). OCT can measure the refractive index distribution, spectral information, polarization information, and the like of an object to be observed by using light having a wide wavelength range as a probe. In addition, it is possible to observe the three-dimensional structure of the object to be observed without staining or noninvasively. Therefore, OCT is suitable for in vivo cells.

On the other hand, there is also a demand for observing a three-dimensional structure of an object to be observed in a non-stained / non-invasive manner using OCT in cultured cells or intracellular organs. However, many observation objects such as cultured cells and intracellular organs described above are generally transparent tissues, and scattered light obtained by backscattering is weak. Further, these objects to be observed are often held in highly reflective containers such as slide glasses and petri dishes. Therefore, the observation object cannot be suitably observed by simply applying the conventional reflective OCT as it is. Further, only by transforming the reflective OCT to the transmissive type, the transmitted light transmitted through the entire object to be observed is detected, and thus the distribution in the depth direction of the object to be observed cannot be measured.

The present invention has been made in view of the above problems, and an object thereof is to provide an observation apparatus and an observation method capable of detection based on light transmitted through an object to be observed.

One aspect of the observation apparatus of the present invention includes a branching unit that branches incident light into reference light and measurement light, an illumination optical system that irradiates the object to be observed along the predetermined direction, and the observation object. An observation optical system that is disposed on a different side from the illumination optical system with respect to an object and receives observation light that travels in a direction different from the predetermined direction among the measurement light via the object to be observed; and the reference light A reference optical system that receives the reference light and the observation light synthesized by the synthesis optical system, and combines the reference light and the observation light. A detection device that detects interference intensity with the observation light.

The branching unit branches light in a predetermined wavelength range into the reference light and the measurement light, and the detection device splits the reference light and the observation light synthesized by the synthesis optical system. An element and a detection element that detects the interference intensity for each of a plurality of different wavelength ranges among the light dispersed by the spectroscopic element may be included.

Further, the detection device may include position information acquisition means for acquiring information related to the position of the observation object along a direction different from the predetermined direction based on the interference intensity for each of the plurality of wavelength ranges.

Further, the detection device may include position information acquisition means for acquiring information related to the position of the observation object along a direction different from the predetermined direction based on the interference intensity.

The observation optical system has an optical axis along a direction different from the predetermined direction, and the illumination optical system transmits the measurement light from a direction inclined by a predetermined angle with respect to the optical axis of the observation optical system. The object to be observed may be irradiated.

The observation optical system has an optical axis along a direction different from the predetermined direction, and the illumination optical system transmits the measurement light from a direction inclined by a predetermined angle with respect to the optical axis of the observation optical system. Irradiating the object to be observed, the position information acquisition means may acquire information on the position of the object to be observed along the optical axis of the observation optical system based on the interference intensity for each of the plurality of wavelength ranges. good.

In addition, the illumination optical system may include shape control means arranged in the optical path of the illumination optical system in order to control the predetermined direction of the measurement light irradiated on the object to be observed.

Further, the illumination optical system may include an oblique incident means that forms an intensity distribution of incident light in a ring shape in a direction along a cross section of the light beam.

Further, the oblique incident means may include an optical element that deflects light incident along the optical axis of the illumination optical system or the observation optical system.

The oblique incidence means may include a light shielding means for shielding a part of the incident light.

The light shielding means may have at least one opening.

Further, the light shielding means may have a plurality of openings dispersed along a substantially circular shape.

The light shielding means may have an opening that transmits light incident along a substantially circular shape.

Further, scanning means for relatively moving the position of the illumination optical system and the object to be observed may be provided.

Further, if the angle formed by the optical axis of the illumination optical system and the measurement light for oblique incidence illumination is θ, and the position of the object to be observed along the optical axis direction of the observation optical system by the interference light is z, The position information L of the object to be observed may be expressed by the formula L = z / (1−cos θ).

A half-wave plate or a quarter-wave plate disposed in the reference light or the measurement light; a first polarizing element for entering light of a predetermined polarization into the branching unit; A second polarizing element that transmits part of the interference light including a polarization direction that is determined based on predetermined polarization; a polarization direction that is transmitted by the first polarizing element; and a polarization that is transmitted by the second polarizing element. Control means for changing the direction at the same time may be provided.

The numerical aperture NA of the measurement light illuminated obliquely with respect to the optical axis of the observation optical system may be 0.4 <NA <0.8.

Further, the numerical aperture NA of the measurement light illuminated obliquely with respect to the optical axis of the observation optical system may be 0.8.

Also, the optical path length difference between the measurement light and the reference light may be shorter than 400 μm.

One aspect of the observation method of the present invention includes branching incident light into reference light and measurement light, irradiating the object to be observed along the predetermined direction with respect to the object to be observed, Receiving observation light that travels (advances) in a direction different from the predetermined direction out of the measurement light via the object to be observed from a side different from the side irradiated with the illumination light, and the reference light and the observation Combining (superimposing) light with each other, and receiving the reference light and the observation light combined with each other, and detecting interference intensity between the reference light and the observation light.

The branching branches light in a predetermined wavelength range into the reference light and the measurement light, and the detection includes splitting the reference light and the observation light combined with each other. , Detecting the interference intensity for each of a plurality of different wavelength ranges among the reference light and the observation light that have been dispersed.

Further, the detecting may include acquiring information related to a position of the observation object along a direction different from the predetermined direction based on the interference intensity for each of the plurality of wavelength ranges.

It is a block diagram of the OCT apparatus of 1st Embodiment. It is a figure explaining the optical path in the OCT apparatus of a 1st embodiment. It is a figure explaining the aperture 13 in the OCT apparatus of 1st Embodiment. It is a block diagram of the OCT apparatus of 2nd Embodiment. It is a figure explaining the zonal mask 22 in the OCT apparatus of 2nd Embodiment. It is a figure explaining the optical path in the OCT apparatus of a 2nd embodiment. It is another figure explaining the annular zone mask 22 in the OCT apparatus of 2nd Embodiment. It is a block diagram of the OCT apparatus in the modification of 1st Embodiment and 2nd Embodiment. It is a block diagram of the OCT apparatus of 3rd Embodiment. It is a figure explaining a beam scan type OCT apparatus. It is another figure explaining a beam scan type OCT apparatus.

[First Embodiment]
The OCT apparatus according to the first embodiment of the present invention will be described below.

FIG. 1 is a configuration diagram of the OCT apparatus according to the first embodiment. As shown in FIG. 1, the OCT apparatus includes a light source 1, a collimating lens 2,

beam splitters

3 and 4, an axicon lens 5, a relay lens 6, total reflection mirrors 7 and 8,

objective lenses

9 and 10, a sample 11, and a sample stage. 12, an aperture 13, a dispersion correction optical member 14, a cylindrical lens 15, a spectrum detector 16, a control device 17, an arithmetic device 18, and the like are disposed. The control device 17 controls each part of the light source 1, the sample stage 12, and the spectrum detector 16 and sends the spectrum signal acquired by the spectrum detector 16 to the arithmetic device 18.

In other words, the observation optical system such as the objective lens 10 is different from the illumination optical system such as the objective lens 9 from the light source 1 with respect to the sample 11 that is the observation object (that is, the observation object is placed on the illumination optical system). To be sandwiched). Then, the observation optical system such as the objective lens 10 receives the observation light that travels (advances) in a direction different from the irradiation direction of the illumination light by the illumination optical system among the measurement light that has passed through the sample 11 that is the object to be observed.

In the sample 11, for example, cultured cells or the like to be observed are cultured in a container (not shown). In addition, as a container mentioned above, various things, such as a petri dish, a flask, a well plate, a microplate, can be used. In addition, a slide glass can be used instead of the container.

Further, the sample 11 is set on the sample stage 12. The sample stage 12 is movable in a plane (xy plane) perpendicular to the optical axis direction of the

objective lenses

9 and 10, and performs scanning in the xy direction when detected by the OCT apparatus (details will be described later).

Hereinafter, the case where the light source 1 emits light having a short time coherence will be described. As the light source 1, for example, a super luminescence diode (SLD), a titanium sapphire laser, a white LED, or the like is applied.

Note that the resolution in the z direction of the OCT apparatus depends on the coherent length of the light source 1. Further, the resolution in the xy direction of the OCT apparatus depends on the size of a condensing point to be described later, and the size of the condensing point depends on the performance of the objective lens 9.

The illumination light L0 emitted from the light source 1 is collimated to a predetermined beam diameter by the collimator lens 2 and enters the beam splitter 3. The illumination light L0 that has entered the beam splitter 3 is branched into a reference light Lr that travels toward the dispersion correction optical member 14 and a measurement light Lm that travels toward the sample 11.

The reference light Lr enters the beam splitter 4 through the dispersion correction optical member 14 and the total reflection mirror 8.

Here, the dispersion correction optical member 14 is an element mainly for maintaining the balance of the interferometer in the OCT apparatus. The balance of the interferometer here refers to the balance of chromatic dispersion of the sample arm and the reference arm of the interferometer. This balance may be lost due to an optical material such as an objective lens and a medium (a culture solution or the like) included in the sample.

Therefore, the dispersion correcting optical member 14 includes a member for correcting the balance loss caused by the optical material such as an objective lens, and a member for correcting the balance loss caused by the medium included in the sample. . The member for correcting the balance loss caused by the optical material described above has a correction amount corresponding to each optical member arranged on the sample arm. In addition, the member for correcting the balance loss caused by the medium described above is preferably a member whose correction amount is variable in accordance with the type and amount of the medium. As an example, a glass block or the like is used as a member for correcting the balance loss caused by the optical material. Further, as a member for correcting the balance loss caused by the medium, an AOPDF (Acousto-Optic programmable dispersive filter), a prism pair that can be inserted and changed, and the like are used. In addition, when the correction amount and the correction content can be corrected by calculation processing, the dispersion correction optical member 14 may be substituted by calculation processing.

The reference light Lr is adjusted by the dispersion correcting optical member 14 so as to have a dispersion balanced with the sample arm. The reference light Lr that has passed through the dispersion correction optical member 14 is hereinafter referred to as reference light Lr ′.

Generally, it is known that the interference pattern (in the spectral direction) becomes finer as the optical path length difference between the measurement light and the reference light in the OCT apparatus increases. And the finer the interference fringes, the more difficult it is to spectrally resolve and detect with a spectroscopic system. That is, the upper limit of the optical path length difference is determined by the resolution of the spectroscopic system (the spectroscope portion).

For example, taking a case where a spectroscopic system having a wavelength resolution of 0.15 nm near 800 nm is used as an example, from the sampling theorem, for example, when the optical path length difference> 400 μm, it becomes difficult to detect interference fringes. Therefore, for example, it is desirable that the optical path length difference <400 μm.

On the other hand, the measurement light Lm is incident on the axicon lens 5, bent at an angle corresponding to the apex angle of the axicon lens 5, and then guided to the relay lens 6.

Here, the axicon lens 5 is a conical lens also called a conical lens, and is used for forming an annular beam (donut shape) or a Bessel beam (non-diffractive-Bessel beam). In general, annular illumination is often introduced for use in extending the depth of focus using a Bessel beam or the like. Although FIG. 1 illustrates a convex axicon lens, a concave axicon lens may be applied. In addition to the axicon lens, any member may be used as long as the measurement light Lm can be changed into an annular shape in the light speed cross section. For example, at least a part of the measurement light Lm may be changed into an annular shape by combining a plurality of mirrors. In the present embodiment, the annular illumination is for obliquely incident measurement light on the sample 11 (details will be described later).

Measurement light (hereinafter referred to as measurement light Lm ′) formed in an annular shape by the axicon lens 5 is guided to the objective lens 9 via the relay lens 6 and the total reflection mirror 7.

In addition, when the light that is formed in an annular shape by the axicon lens 5 and can be brought to the pupil position by the objective lens 9 is referred to as “annular pattern”, numerical examples of the annular pattern are as follows.

Suppose, for example, that a high NA is advantageous in favor of Z resolution, and an objective lens with NA 0.8 is used in consideration of feasibility. At this time, from NA = 0.8 = sin θ, θ = 54 °. For example, if the coherence length of the light source 1 at this time is 2 μm, the Z resolution is ˜10 μm. At this time, if the zone width of the above-described zone pattern is 200 μm, the depth of focus is about 200 / 0.8 mm to 250 μm.

That is, when NA = 0.8 (θ = 54 °) and the bandwidth is 200 μm, the Z resolution (10 μm) and the depth of focus (250 μm) are balanced. For example, if the observation target is a living body, This is particularly effective because it is suitable for structural observation.

The measurement light Lm ′ from the total reflection mirror 7 is condensed by the objective lens 9 and irradiated toward one point (condensing point) in the deep part of the sample 11. Note that the relative position in the z direction between the sample 11 and the objective lens 9 is adjusted in advance so that the focal plane of the objective lens 11 covers the region where the object to be observed (such as cultured cells) in the sample 11 is present. Further, at least one pupil (Fourier transform plane) of the illumination surface in the sample 11 exists between the relay lens 6 and the sample 11. The pupil position is indicated by Fa in FIG. This pupil position is also the focal plane of the objective lens 9. The measurement light Lm ′ is condensed on the focal plane to form a point light source (secondary light source), so that the sample 11 is illuminated with a parallel wave (collimated light).

In the sample 11 in the irradiation region of the measurement light Lm ′ (hereinafter referred to as “irradiation spot”), diffracted light with various angles may be generated. Of these diffracted lights, the light traveling in the same direction as the measurement light Lm ′ toward the condensing point is captured by the objective lens 10 through the aperture 13.

The specification of the objective lens 10 is the same as the specification of the objective lens 9 described above, and the arrangement destination of the objective lens 10 is a position facing the objective lens 9 with the sample 11 interposed therebetween. The focal plane of the objective lens 10 coincides with the focal plane of the objective lens 9, and the focal point of the objective lens 10 coincides with the focal point of the objective lens 9.

Hereinafter, of the measurement light traveling from the irradiation spot toward the objective lens 10, the light captured by the objective lens 10 is referred to as “measurement light Lm”. The measurement light Lm ″ passes through the objective lens 10 and then enters the beam splitter 4.

Here, details from the illumination light L0 emitted from the light source 1 to the measurement light Lm ″ incident on the beam splitter 4 will be described with reference to FIG.

2 is an optical path diagram from the illumination light L0 emitted from the light source 1 to the measurement light Lm ″ incident on the beam splitter 4. As shown in FIG. 2, the illumination light L0 emitted from the light source 1 is a collimating lens. 2 is collimated to a predetermined beam diameter by 2. Then, the measurement light Lm is formed in an annular shape by the axicon lens 5. Further, the measurement light Lm ′ having passed through the relay lens 6 is irradiated onto the sample 11 by the objective lens 9. At this time, the measurement light Lm ′ is obliquely incident on the sample 11. In the sample 11, diffracted light is generated according to the contents of the observation object of the sample 11. This diffracted light is used as the objective. It is captured by the lens 10. However, at this time, the aperture 13 blocks the 0th-order diffracted light component, as shown in FIG. It is a stop for mainly taking out the scattered component by the sample 11 among the measurement light which has been arrange | positioned between the objective lenses 10 and permeate | transmitted the sample 11. Moreover, the aperture 13 is the aperture 13 between the objective lens 10 and the beam splitter 4. In this case, it is preferable that the aperture 13 is disposed on the light condensing surface (Fourier transform surface) of the objective lens 10 on which the 0th-order diffracted light component transmitted through the sample 11 is condensed. .

FIG. 3 shows an enlarged view of the vicinity of the aperture 13. As shown in FIG. 3, the aperture 13 has a predetermined aperture, and transmits only the diffracted light near the center (shaded portion in FIG. 3) among the diffracted light by the sample 11, and blocks the other light. .

Then, the measurement light Lm ″ incident on the beam splitter 4 is integrated with the reference light Lr ′ incident on the beam splitter 4 from the reference arm side and travels toward the cylindrical lens 15. The integrated reference light Lr. 'And measurement light Lm' are collectively referred to as "interference light". The interference light directed toward the cylindrical lens 15 is guided by the cylindrical lens 15 to the entrance slit of the spectrum detector 16.

Here, the interference light guided to the entrance slit of the spectrum detector 16 includes various light different from the light illuminated by the illumination optical system such as the light source 1 and the objective lens 9 (transmitted through the sample 11). Light, light reflected by the sample 11, and light diffracted by the sample 11.

As shown in FIG. 1, the spectrum detector 16 includes a slit plate 16a having a slit opening at a condensing point of the interference light, a collimator mirror 16b for converting the interference light that has passed through the slit plate 16a into parallel light, A reflective diffraction grating 16c that separates the interference light that has become parallel light into a plurality of wavelength components, a condensing mirror 16d that condenses the wavelength components at positions shifted from each other, and each of the light that is condensed at positions shifted from each other. And a line sensor 16e for individually detecting the intensity of the wavelength component. With this configuration, the spectrum detector 16 generates an intensity signal (that is, a spectrum signal) for each wavelength component of the interference light. This spectrum signal is sent to the control device 17.

Here, the sample stage 12 described above can displace the sample 11 in the xy direction under the control of the control device 17. Therefore, when the sample stage 12 is driven, the irradiation spot on the sample 11 moves in the xy direction. Note that the form of spectral spectroscopy and detection of interference light is not limited to FIG.

Therefore, the control device 17 drives the sample stage 12 to perform two-dimensional scanning on the sample 11 in the xy direction with the irradiation spot, and when the irradiation spot is at each xy position, drives the line sensor 16e to obtain the spectrum signal. By taking in, the spectrum signal of each xy position is acquired. These spectrum signals are sent to the arithmetic unit 18.

The arithmetic unit 18 obtains structural information in the z direction at each xy position by individually Fourier transforming the spectrum signal at each xy position. Thereby, the three-dimensional image information in the xyz direction becomes known (details will be described later). The arithmetic unit 18 displays the structural information of the sample 11 that has become known on a monitor (not shown).

As described above, according to the present embodiment, the measurement light guided from the branching unit is obliquely incident on the object to be observed by using the axicon lens, and the reference light transmitted through the reference object and the object to be measured. The measurement light transmitted through the observation object is combined, and the intensity of the combined light composed of the combined reference light and measurement light is detected. Therefore, it is possible to detect the internal structure of the observation object based on the light transmitted through the observation object.

In particular, according to the present embodiment, even when the object to be observed is a transparent tissue or when the object to be observed is held in a highly reflective container, it is possible to perform suitable observation by OCT. it can.

[Second Embodiment]
The OCT apparatus according to the second embodiment of the present invention will be described below.

FIG. 4 is a configuration diagram of the OCT apparatus according to the second embodiment. 4, the same elements as those shown in FIG. 1 are denoted by the same reference numerals. As shown in FIG. 4, in the OCT apparatus of this embodiment, the axicon lens 5 and the relay lens 6 shown in FIG. 1 are omitted, and a beam expander 21 and an annular mask 22 are arranged instead.

The beam expander 21 is disposed between the beam splitter 3 and the total reflection mirror 7 and converts the beam diameter. If the beam diameter of the light emitted from the light source 1 is sufficiently wide, the beam expander 21 need not be used.

The annular mask 22 is arranged at the pupil position of the objective lens 9 (the pupil position on the side opposite to the sample 11), guided from the beam splitter 3, and the measurement light Lm whose beam diameter is converted by the beam expander 21. Shield part of the light. As shown in FIG. 5, the annular zone mask 22 is a mask having an annular opening, and the annular zone diameter is d. An annular pattern having a bandwidth ρ is formed at the pupil position of the objective lens 9 due to the balance between the aperture band width and the distance from the annular mask 22 to the pupil position of the objective lens 9. At this time, the relationship of the following equation holds for the depth of focus.

Depth of focus = 2ρf / D˜ρ / NA (Formula 1)
In Equation 1, f represents the focal length of the objective lens 9, and NA represents the effective NA of the objective lens.

Further, numerical examples of the annular pattern by the annular mask 22 are as follows.

Suppose, for example, that a high NA is advantageous in favor of Z resolution, and an objective lens with NA 0.8 is used in consideration of feasibility. At this time, from NA = 0.8 = sin θ, θ = 54 °. For example, if the coherence length of the light source 1 at this time is 2 μm, the Z resolution is ˜10 μm. At this time, if the band width ρ of the above-described annular pattern is 200 μm, the depth of focus is about 200 / 0.8 mm to 250 μm.

That is, when NA = 0.8 (θ = 54 °) and the bandwidth ρ = 200 μm, the Z resolution (10 μm) and the depth of focus (250 μm) are balanced. For example, when the observation target is a living body, This is particularly effective because the specifications are appropriate for the structure observation.

6 is an optical path diagram from the illumination light L0 emitted from the light source 1 to the measurement light Lm ″ incident on the beam splitter 4. As shown in FIG. 6, the illumination light L0 emitted from the light source 1 is a collimating lens. The measurement light Lm is converted to a predetermined beam diameter by the beam expander 21. Further, the measurement light Lm that has passed through the total reflection mirror 7 is applied to the annular mask 22. The measurement light Lm is formed in an annular shape by the annular mask 22, and the measurement light Lm ′ formed in an annular shape is irradiated onto the sample 11 by the objective lens 9. At this time, the measurement light Lm ′ is In the same manner as in the first embodiment, the incident light is obliquely incident on the sample 11. In the sample 11, diffracted light is generated according to the contents of the observation object of the sample 11. The component that passes through the sample 11 is captured by the objective lens 10. However, at this time, the aperture 13 blocks part of the transmitted light (0th-order diffracted light component). This is the same as in the first embodiment.

Then, the measurement light Lm ″ incident on the beam splitter 4 is integrated with the reference light Lr ′ incident on the beam splitter 4 from the reference arm side toward the cylindrical lens 15. The subsequent cylindrical lens 15 and spectrum detection are performed. The configurations and operations of the device 16, the control device 17, and the calculation device 18 are the same as those in the first embodiment.

As described above, according to the present embodiment, the measurement light guided from the branching unit is obliquely incident on the object to be observed by using the annular mask, and the reference light transmitted through the reference object and the object to be observed. The measurement light transmitted through the observation object is combined, and the intensity of the interference component contained in the combined light composed of the combined reference light and measurement light is detected. Therefore, the same effect as that of the first embodiment can be obtained.

[Supplement of the second embodiment]
In the OCT apparatus of the second embodiment, an example in which oblique incidence is realized using the annular mask 22 shown in FIG. 5 has been shown, but the present invention is not limited to this example.

For example, a mask having a single opening may be used instead of the annular zone mask 22. As shown in FIG. 7A, in the case of a mask having a pinhole at one point corresponding to the x-axis shape, it has resolution in the x and z directions. By adding a mask having one opening for one round around the optical axis of the objective lens 9, information equivalent to that when the annular mask 22 is arranged can be obtained. This method can reduce dark noise and make it easier to obtain an S / N ratio when a low-intensity light source is used.

Also, for example, a mask having a plurality of discrete or continuous openings on a substantially constant circle may be used instead of the annular mask 22. In addition, as shown in FIG. 7B, a mask having pinholes at one point corresponding to the x-axis and one point corresponding to the y-axis has a three-dimensional resolution.

Furthermore, as shown in FIG. 7C, in the case of a mask having pinholes at two symmetrical points corresponding to the x-axis and two symmetrical points corresponding to the y-axis, the mask has three-dimensional resolution. Furthermore, distortion of PSF can be suppressed and the depth of focus can be increased.

As described above, 2π / 4N (where N = 1, 2,...) Openings are formed in an annular shape, and the larger N is set, the more suitable the zonal mask can be made. The openings do not necessarily have to be arranged symmetrically. Further, the shapes of the plurality of openings are not necessarily the same.

[Modifications of First Embodiment and Second Embodiment]
The control device 17 controls the objective lens 9 and the annular mask 22 in conjunction with each other according to the sample 11. By such control, it becomes possible to irradiate measurement light from various directions to the object to be observed included in the sample 11, and to capture the three-dimensional structure evenly.

In this way, the numerical aperture is variable, the objective lens that condenses the measurement light guided from the branching unit on the object to be observed is provided, and the numerical aperture of the objective lens is determined according to the content of detection by the detecting unit. To control. Therefore, suitable detection according to the object to be observed can be made possible.

In particular, even when the object to be observed is a transparent tissue or when the object to be observed is held in a highly reflective container, suitable observation by OCT can be performed.

Further, by controlling the annular zone diameter of the annular mask in conjunction with the numerical aperture of the objective lens described above, it is possible to detect more precisely according to the object to be observed.

Also, in actual detection, the resolution and depth of focus required for the OCT apparatus differ depending on what structure of what observation object is desired to be detected. For example, when an object to be observed is an observation object of a relatively large scale such as a cell spheroid, and it is desired to observe an overview of the object, a resolution and a depth of focus on the order of the spheroid size are required. In addition, when the object to be observed is a minute object such as a cell nucleus and it is desired to observe its detailed structure, high resolution is required even at the expense of the depth of focus. According to the processing as described above, the resolution can be appropriately changed according to the scale of the object to be observed.

In any case, if the light that is formed in an annular shape by an axicon lens or an annular mask and can be brought to the pupil position by the objective lens 9 is called an “annular pattern”, numerical examples of the annular pattern are as follows. .

For example, assuming that the coherence length of the light source 1 is 2 μm, NA is 0.4 <NA <0.8 mm, and the band width of the annular pattern is 200 μm, NA = sinθ, and the range of θ is 6 ° <θ <55 ° It becomes. The Z resolution at this time is 10 mm (μm) <Z resolution <50 mm (μm), and the focal depth is 250 mm (μm) <focus depth <500 mm (μm). In other words, when 0.4 <NA <0.8 and the bandwidth is 200 μm, the Z resolution is 10 (μm) to 50 mm (μm) and the depth of focus is 250 (μm) to 500 mm (μm). When the object is a living body, it is particularly effective because it is a specification suitable for structure observation.

Note that oblique incidence illumination may be applied to a reflection type OCT assumption. Specific examples are shown below.

FIG. 8 is a configuration diagram of an OCT apparatus according to this modification. In FIG. 8, the same elements as those shown in FIG. As shown in FIG. 8, the OCT apparatus includes a light source 1, a beam splitter 3, an objective lens 9, a sample 11, a cylindrical lens 15, a spectrum detector 16, a control device 17, a calculation device 18 and the like similar to those in the first embodiment. Thus, a plane mirror (reference mirror) 41 and an optical scanner 42 are arranged.

The illumination light L0 emitted from the light source 1 enters the beam splitter 3 and is branched into a reference light Lr traveling toward the reference mirror 41 and a measurement light Lm traveling toward the sample 11.

When the reference light Lr is incident on the reference mirror 41 from the front, the reference light Lr is reflected by the reference mirror 41 and returns to the beam splitter 3.

On the other hand, when the measurement light Lm is incident on the objective lens 9 via the optical scanner 42, the measurement light Lm receives the light condensing action of the objective lens 9 and is condensed toward one point (condensing point) in the deep part of the sample 11. Note that the relative position in the z direction between the sample 11 and the objective lens 9 is adjusted in advance so that the focal plane of the objective lens 9 is applied to a region where an object to be observed (such as cultured cells) in the sample 11 is present.

In the irradiation area of the measurement light Lm in the sample 11 (hereinafter referred to as “irradiation spot”), reflected light of various angles may be generated. Of the reflected light, the light that follows the optical path of the measurement light Lm toward the condensing point in the opposite direction is captured by the objective lens 9. Hereinafter, the light captured by the objective lens 9 out of the reflected light from the irradiation spot toward the objective lens 9 will be referred to as “measurement light Lm ′”. The measurement light Lm ′ follows the optical path of the measurement light Lm in the reverse direction and enters the beam splitter 3 via the optical scanner 42.

The measurement light Lm ′ incident on the beam splitter 3 is integrated with the reference light Lr ′ returned to the beam splitter 3 and the optical path, and travels toward the cylindrical lens 15. Here, the optical path length of the single optical path of the reference light (optical path length of the reference arm) and the optical path length of the single optical path of the measurement light (optical path length of the measurement arm) coincide with each other.

The integrated reference light Lr ′ and measurement light Lm ″ are collectively referred to as “interference light”. The interference light directed toward the cylindrical lens 15 is guided by the cylindrical lens 15 to the entrance slit of the spectrum detector 16.

The configuration and operation of the spectrum detector 16 are the same as those in the first embodiment.

Here, when the above-described optical scanner 42 is driven, the above-described condensing point moves in the field of view of the objective lens 9, so that the irradiation spot on the sample 11 moves in the xy direction.

Accordingly, the control device 17 drives the optical scanner 42 to perform two-dimensional scanning on the sample 11 in the xy direction with the irradiation spot, and when the irradiation spot is at each xy position, drives the line sensor 16e to output the spectrum signal. By taking in, the spectrum signal of each xy position is acquired. These spectrum signals are sent to the arithmetic unit 18.

The arithmetic unit 18 obtains structural information in the z direction at each xy position by individually Fourier transforming the spectrum signal at each xy position. As a result, the cell distribution in the xyz direction becomes known. The arithmetic unit 18 displays the distribution of the structural information in the sample 11 that has become known on a monitor (not shown).

The control device 17 controls the numerical aperture NA of the objective lens 9 according to the sample 11. By such control, it becomes possible to irradiate measurement light from various directions to the object to be observed included in the sample 11, and to capture the three-dimensional structure evenly.

As described above, according to the present modification, the same effects as those of the above-described embodiments can be obtained even in the reflective OCT apparatus.

[Third Embodiment]
The OCT apparatus according to the third embodiment of the present invention will be described below.

FIG. 9 is a configuration diagram of the OCT apparatus of the third embodiment. In FIG. 9, the same elements as those shown in FIG. As shown in FIG. 9, in the OCT apparatus of this embodiment, the axicon lens 5 and the relay lens 6 shown in FIG. 1 are omitted, and an optical scanner 31 is arranged instead.

The optical scanner 31 includes two total reflection mirrors 31a and 31b, and changes the incident angle of the measurement light Lm to the objective lens 9. The optical scanner 31 is controlled by the control device 17.

The controller 17 controls the optical scanner 31, the total reflection mirror 7, and the total reflection mirror 8 in conjunction with each other so that the measurement light Lm ′ is obliquely incident on the sample 11. At this time, for example, if the irradiation spot that is obliquely incident is rotated and the irradiation is performed in a ring shape, substantially the same detection as in the first embodiment and the second embodiment described above can be performed. Such a method can be expected to reduce dark noise and easily obtain an SN ratio when a low-intensity light source is used as the light source.

The measurement light Lm ′ is obliquely incident on the sample 11 by the above-described optical scanner 31 as in the first embodiment. The configuration and operation of the subsequent aperture 13, beam splitter 4, cylindrical lens 15, spectrum detector 16, control device 17, and calculation device 18 are the same as those in the first embodiment.

As described above, according to the present embodiment, the measurement light guided from the branching unit is obliquely incident on the object to be observed by using the optical scan including the annular mirror, and is transmitted through the reference object. The reference light and the measurement light transmitted through the object to be observed are combined, and the intensity of the combined light composed of the combined reference light and measurement light is detected. Therefore, the same effect as that of the first embodiment can be obtained.

[Supplement of the third embodiment]
In the OCT apparatus according to the third embodiment, instead of performing scanning on the xy plane by the sample stage 12, scanning may be performed by the optical scanner 31. Two scanning methods may be selectively used or may be used in combination.

In the OCT apparatus of the third embodiment, an example in which the oblique incidence is realized by the optical scanner 31 has been shown, but the present invention is not limited to this example. For example, oblique incidence may be realized by electrical control using a micro device mirror, a DOE (high precision diffractive optical element), a spatial modulation element such as liquid crystal, and the like. Furthermore, as described in the first embodiment and the second embodiment, the measurement light may be formed in an annular shape using the spatial modulation element described above.

[Modification of Embodiment]
The OCT apparatus of each embodiment described above employs a method of displacing the sample 11 side by moving the sample stage 12 (stage scan type), but a method of displacing the irradiation spot side (beam scan type). May be adopted. Two methods may be selectively used or may be used in combination.

FIG. 10 is a configuration diagram of a part of a beam scan type OCT apparatus. In FIG. 10, the part corresponding to between the total reflection mirror 7 and the beam splitter 4 in FIG. 4 is illustrated. As shown in FIG. 10, in this modification, the sample stage 12 is omitted, and each part of the total reflection mirror 51, the mirror pairs 52a and 52b, the mirror pairs 53a and 53b, and the total reflection mirror 54 is provided. The objective lens 9, the objective lens 10, and the aperture 13 are fixed. Further, the

mirror pair

52a and 52b are arranged so that the center of the

mirror pair

52a and 52b is the pupil position of the objective lens 9 (Fa in FIG. 10), and the center of the

mirror pair

53a and 53b is the pupil position of the objective lens 10 (Fb in FIG. 10). ). And the control apparatus 17 scans by controlling each part of

mirror pair

52a and 52b and

mirror pair

53a and 53b synchronously. The

mirror pair

52a and 52b has a three-dimensional structure as shown in FIG. 11, and emits light with an incident light velocity bent by 90 degrees. The same applies to the mirror pairs 53a and 53b.

Moreover, although the OCT apparatus of each embodiment mentioned above employ | adopted the method (Fourier domain type) which carries out the spectral detection of the white interference light using a light source (white light source), it scans a light source wavelength and interferes with each wavelength. A method of detecting light in a time division manner (wavelength scanning type) may be employed.

Incidentally, when the wavelength scanning type is adopted, it is not necessary to perform spectral detection, so by using an image sensor instead of the spectrum detector 18, the interference light intensity at each position in the xy direction on the sample 11 can be collectively displayed. It may be detected.

Moreover, although the OCT apparatus of each embodiment mentioned above employ | adopted the method (Fourier domain type) which carries out the spectral detection of the interference light using a light source (white light source), it uses a light source (white light source), and is measured. You may employ | adopt the method (time domain type) which scans the optical path length difference of light and reference light, and detects white interference light for every scanning position.

Incidentally, when the time domain type is adopted, it is not necessary to perform spectral detection, so by using an image sensor instead of the spectrum detector 18, the interference light intensity at each position in the xy direction on the sample 11 is collectively displayed. It may be detected.

Further, in each of the above-described embodiments, an example in which illumination light is obliquely incident on an object to be observed by controlling (adjusting) the illumination optical system has been described, but the present invention is not limited to this example. For example, even when illumination light is incident vertically on the object to be observed, the same effect as when obliquely incident illumination light is obtained by performing control (adjustment) to tilt the sample stage of the sample that is the object to be observed. Can be obtained. Furthermore, the control (adjustment) of obliquely incident illumination light on the object to be observed and the control (adjustment) of tilting the sample stage of the sample that is the object to be observed may be performed in combination.

Further, in the OCT apparatus of each embodiment described above, a polarization component may be used. For example, a difference is provided between the polarization direction of the reference light toward the detection means and the polarization direction of the measurement light toward the object to be observed, and interference light is included from the combined light of the reference light and measurement light toward the detection means. Detection using the polarization component can be performed by transmitting the polarization component in the direction and removing the polarization component in the direction not including the interference light.

DESCRIPTION OF SYMBOLS 1 ... Light source, 3, 4 beam splitter, 5 ... Axicon lens, 9, 10 ... Objective lens, 11 ... Sample, 13 ... Aperture, 14 ... Optical member for dispersion correction, 16 ... Spectrum detector, 17 ... Control apparatus, 18 ... arithmetic unit, 22 ... annular mask, 31 ... optical scanner

Claims

A branching means for branching the incident light into reference light and measurement light;
An illumination optical system for irradiating the object to be observed along the predetermined direction with the measurement light;
An observation optical system that is disposed on a different side from the illumination optical system with respect to the object to be observed, and that receives observation light traveling in a direction different from the predetermined direction among the measurement light via the object to be observed;
A combining optical system that guides the reference light to the observation optical system and combines the reference light and the observation light;
A detection device that receives the reference light and the observation light synthesized by the synthesis optical system and detects an interference intensity between the reference light and the observation light;
An observation apparatus comprising:
The observation apparatus according to claim 1,
The branching unit branches light in a predetermined wavelength range into the reference light and the measurement light,
The detection device includes:
A spectroscopic element for dispersing the reference light and the observation light synthesized by the synthesis optical system;
An observation apparatus comprising: a detection element that detects the interference intensity for each of a plurality of different wavelength ranges among light dispersed by the spectral element.
The observation apparatus according to claim 2,
The detection device includes position information acquisition means for acquiring information related to the position of the object along a direction different from the predetermined direction based on the interference intensity for each of the plurality of wavelength regions. apparatus.
The observation apparatus according to claim 1 or 2,
The observation apparatus includes position information acquisition means for acquiring information related to the position of the observation object along a direction different from the predetermined direction based on the interference intensity.
In the observation apparatus according to any one of claims 1 to 4,
The observation optical system has an optical axis along a direction different from the predetermined direction,
The illuminating optical system irradiates the object to be observed with the measurement light from a direction inclined by a predetermined angle with respect to the optical axis of the observation optical system.
The observation device according to claim 3,
The observation optical system has an optical axis along a direction different from the predetermined direction,
The illumination optical system irradiates the object to be observed with the measurement light from a direction inclined by a predetermined angle with respect to the optical axis of the observation optical system,
The observation apparatus characterized in that the position information acquisition means acquires information related to the position of the object to be observed along the optical axis of the observation optical system based on the interference intensity for each of the plurality of wavelength regions.
In the observation apparatus according to any one of claims 1 to 6,
The illumination optical system includes:
An observation apparatus comprising: shape control means arranged in an optical path of an illumination optical system in order to control the predetermined direction of the measurement light applied to the object to be observed.
The observation apparatus according to any one of claims 1 to 7,
The illumination optical system includes:
An observation apparatus comprising oblique incidence means for forming an intensity distribution of incident light in a ring shape in a direction along a cross section of the light beam.
The observation apparatus according to claim 8, wherein
The oblique incident means includes an optical element that deflects light incident along the optical axis of the illumination optical system or the observation optical system.
The observation device according to claim 8 or 9,
The oblique incident means includes a light shielding means for shielding a part of the incident light.
The observation device according to claim 10,
The light shielding means has at least one opening.
The observation device according to claim 10 or 11,
The light-shielding means has a plurality of openings dispersed along a substantially circular shape.
The observation device according to claim 10 to 12,
The light shielding means has an opening that transmits light incident along a substantially circular shape.
The observation apparatus according to any one of claims 1 to 13,
An observation apparatus comprising: a scanning unit that relatively moves positions of the illumination optical system and the object to be observed.
The observation apparatus according to any one of claims 1 to 14,
The angle formed by the optical axis of the illumination optical system and the measurement light for oblique incidence illumination is θ,
When the position of the object to be observed along the optical axis direction of the observation optical system due to the interference light is z,
The position information L of the object to be observed is represented by the following expression.
L = z / (1-cosθ)
The interference device according to any one of claims 1 to 15,
A half-wave plate or a quarter-wave plate disposed in the reference light or the measurement light;
A first polarizing element for entering light of predetermined polarization into the branching means;
A second polarizing element that transmits a part of the interference light including a polarization direction determined based on the predetermined polarization;
An observation apparatus comprising: a control unit that simultaneously changes a polarization direction transmitted by the first polarization element and a polarization direction transmitted by the second polarization element.
The observation apparatus according to any one of claims 1 to 16,
An observation apparatus, wherein a numerical aperture NA of the measurement light illuminated obliquely with respect to the optical axis of the observation optical system is 0.4 <NA <0.8.
The observation apparatus according to any one of claims 1 to 17,
An observation apparatus, wherein a numerical aperture NA of the measurement light illuminated obliquely with respect to the optical axis of the observation optical system is 0.8.
The observation apparatus according to any one of claims 1 to 18,
An observation apparatus, wherein an optical path length difference between the measurement light and the reference light is shorter than 400 μm.
Branching incident light into reference light and measurement light;
Irradiating the object to be observed along a predetermined direction with the measurement light;
Receiving observation light that is directed to a direction different from the predetermined direction among the measurement light via the observation object from a side different from a side that irradiates the illumination light to the observation object;
Combining the reference light and the observation light;
Receiving the reference light and the observation light combined with each other, detecting an interference intensity between the reference light and the observation light;
An observation method comprising:
The observation method according to claim 20,
The branching branches light in a predetermined wavelength range into the reference light and the measurement light,
The detecting is
Separating the reference light and the observation light synthesized from each other;
Detecting the interference intensity for each of a plurality of different wavelength ranges among the split reference light and observation light.
The observation method according to claim 21, wherein
The detecting method includes obtaining information on a position of the object to be observed along a direction different from the predetermined direction based on the interference intensity for each of the plurality of wavelength regions.