JP2016145874A - Observation apparatus, calculation method and calculation unit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an observation apparatus advantageous for visualization of phase distribution in a sample.SOLUTION: The observation apparatus includes: an image forming optical system (104) that forms an image of a sample; a modulation part (105) which is disposed in the image forming optical system and which has a first modulation area for modulating a beam of incoming light; imaging means (106) for picking up an image of the sample via the image forming optical system; and calculation means (107) that calculates the distribution of phase difference amount on the sample based on a piece of image information acquired by the imaging means and diffraction light component in the diffraction light from the sample, which is modulated by the first modulation area.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an observation apparatus, and more particularly to a phase difference observation apparatus for observing a phase object.

  A phase contrast microscope or a dark field microscope is used as a method for observing a phase object (sample) typified by unstained cells. A general phase contrast microscope has a ring-shaped opening in the illumination system, and gives a phase difference of π / 2 to the direct light after illuminating the sample in the complex amplitude modulation region arranged in the objective lens. Thus, an image with high contrast can be obtained by interference between direct light and diffracted light. However, when a sample with a large amount of phase difference is observed in such a phase contrast microscope, not only direct light but also low-order diffracted light passes through the complex amplitude modulation region, and an artifact called halo is formed at the boundary between the background and the sample. As a result, the phase distribution could not be visualized quantitatively. A general dark field microscope illuminates with a large numerical aperture so that direct light from an illumination system does not enter the objective lens, and an image is obtained only by diffracted light from a phase object. However, since the conventional dark field microscope forms an image only with high-order diffracted light, an image in which only the boundary of the phase object is emphasized is formed, and only qualitative observation can be performed.

  Patent Documents 1 and 2 disclose methods for reducing such influence and quantitatively visualizing the phase distribution. In Patent Document 1, a point light source is used, and a minute complex amplitude modulation region is arranged in the objective lens so that only direct light is transmitted through the complex amplitude modulation region. The obtained image is expressed by a predetermined formula. The phase distribution is visualized quantitatively by calculation. Further, in Patent Document 2, the phase distribution of the complex amplitude modulation region arranged in the objective lens is made variable, and the phase distribution is quantitatively visualized by a fringe scanning method by acquiring a plurality of images.

Patent No. 20325570 Patent No. 3790905

  However, since only the point light source is assumed in the method disclosed in Patent Document 1, there is a problem that the resolution is reduced and speckles are generated. Further, in the method disclosed in Patent Document 2, since the complex amplitude modulation region has an annular shape similar to that of a general phase contrast microscope, halo is generated, and deconvolution processing is performed to remove this influence. . For this reason, there is a problem that the calculation cost is high, and since it is premised that the image is captured three times or more, it is difficult to image at high speed.

  In view of the above problems, an object of the present invention is to provide an observation apparatus, a calculation method, and a calculation apparatus that are advantageous for visualizing the phase distribution of a sample.

  An observation apparatus according to one aspect of the present invention includes an imaging optical system that forms an image of a sample, a modulation unit that is provided in the imaging optical system and that has a first modulation region that modulates incident light, and the connection. Based on an imaging unit that images the sample through an image optical system, image information obtained by the imaging unit, and a diffracted light component modulated by the first modulation region of the diffracted light from the sample And calculating means for calculating a phase difference amount distribution of the sample.

  Other objects and features of the present invention are illustrated in the following examples.

  According to the present invention, it is possible to provide an observation apparatus, a calculation method, and a calculation apparatus that are advantageous for visualizing the phase distribution of a sample.

It is the schematic of the phase difference observation apparatus in the Example of this invention. It is the figure which showed the complex amplitude modulation area | region CAM and opening IA in the Example of this invention. It is explanatory drawing of the magnitude | size of the complex amplitude modulation area | region CAM in the Example of this invention. It is the graph which showed the image intensity when changing the ratio of the complex amplitude modulation area | region CAM which occupies a pupil. It is explanatory drawing of the phase object for simulation. It is a simulation result of the conventional phase-contrast microscope. 3 is a simulation result of the phase difference observation apparatus of Example 1. FIG. 10 is a simulation result of the phase difference observation apparatus of Example 2. 10 is a simulation result of the phase difference observation apparatus of Example 3. FIG. 10 is a simulation result of the phase difference observation apparatus of Example 4.

  Hereinafter, the phase difference observation apparatus 10 (phase difference microscope, dark field microscope, etc.) of the present invention will be described with reference to FIG. Here, FIG. 1 is a schematic diagram of the phase difference observation apparatus 10 of the present embodiment. Reference numeral 100 denotes a light source. Reference numeral 101 denotes an illumination optical system that guides light from the light source 100 to an irradiated surface (sample disposed on the irradiated surface) 103. Reference numeral 102 denotes an aperture stop provided in the illumination optical system 101 and having an aperture IA. The aperture stop 102 is disposed, for example, at the pupil position (near) in the illumination optical system 101. The aperture IA formed in the aperture stop 102 limits the light emitted from the light source 100. Thereby, the light emitted from the light source 100 is limited by the opening IA and illuminates the sample 103. In the present embodiment, a variable aperture stop is preferably used as the aperture stop 102. By using a variable aperture stop, the contrast and resolution can be changed. Further, visible light or near infrared light (for example, wavelength 400 nm to 1100 nm) is used as light used at this time, and light from the sample 103 passes through the modulation unit 105 disposed in the objective lens 104 and enters the image sensor 106. Form an image. The objective lens 104 functions as an imaging optical system that forms (images) the image of the sample 103 disposed on the irradiated surface on the imaging surface of the imaging element 106. The modulation unit 105 is provided inside the objective lens 104 (for example, arranged at the pupil position (near) of the objective lens 104), and has a complex amplitude modulation region (first modulation region) CAM that modulates incident light. The imaging element 106 functions as an imaging unit that receives an image of the sample 103 (that is, images the sample 103 via the objective lens 104). The image sensor 106 is disposed on the imaging surface of the objective lens 104 (imaging optical system). In addition, an image processing system 107 that generates image information from data obtained by the image sensor 106 and calculates a phase difference amount distribution of the sample 103 is provided. The image processing system 107 functions as a calculation unit that calculates the phase difference amount distribution of the sample 103 based on image information of the sample obtained by imaging via the modulation unit 105 in the objective lens 104. As described above, the image processing system 107 includes generation means (image processing apparatus) that generates image information from data obtained by the image sensor 106 and calculation means (calculation apparatus) that calculates the phase difference amount distribution of the sample 103. It is composed.

  Next, the configuration of the aperture stop 102 and the modulation unit 105 used in the phase difference observation apparatus 10 will be described with reference to FIG. The modulation unit 105 is disposed at a position conjugate with the opening IA formed in the aperture stop 102. The modulation unit 105 includes a complex amplitude modulation region CAM (first modulation region) and an amplitude modulation region AM (second modulation region different from the first modulation region) different from the complex amplitude modulation region CAM. The complex amplitude modulation region CAM is arranged so that direct light from the light source 100 that has passed through the aperture IA is incident thereon. The amplitude modulation area AM is arranged so that direct light from the light source 100 that has passed through the opening IA does not enter. FIG. 2 is a configuration diagram of the aperture stop 102 and the modulation unit 105. 2A to 2D are diagrams in which the complex amplitude modulation area CAM and the amplitude modulation area AM of the modulation unit 105 arranged in the objective lens 104 are viewed from the direction of the optical axis AX. . 2A to 2D are diagrams in which the aperture IA of the aperture stop 102 provided in the illumination optical system 101 is viewed from the direction of the optical axis AX.

  2A is a radial shape, FIG. 2B is a spiral shape, FIG. 2C is a multiple ring zone, FIG. 2D is a complex amplitude modulation area CAM of random dots and an amplitude modulation area AM surrounding them. Each opening IA is shown as an example of this embodiment. The complex amplitude modulation region CAM has a shape that is the same as or similar to (similar to) the opening IA, or a shape that is similar to the shape of the opening IA. That is, the number of complex amplitude modulation regions CAM is formed to be the same as the number of openings IA or more than the number of openings IA. However, the present embodiment is not limited to these. For example, the four examples shown in FIGS. 2A to 2D can be combined.

  The phase difference observation apparatus 10 of the present embodiment needs to be configured so that direct light from the aperture IA enters the complex amplitude modulation region CAM. For this reason, as shown in FIG. 2, the ratio of the aperture to the pupil (pupil region) is larger in the complex amplitude modulation region CAM than the aperture IA, or the same (equivalent) size. It is configured. The complex amplitude modulation area CAM is configured to modulate the phase and intensity of direct light from the aperture IA, and the amplitude modulation area AM is configured to modulate the intensity of diffracted light. At this time, the phase modulation amount of the complex amplitude modulation area CAM is determined based on the amplitude modulation area AM. For example, the complex amplitude modulation region CAM is configured to give a phase difference by film formation or cutting on a parallel plate, or to reduce light, block light, or the like (or a combination thereof).

  Preferably, from the viewpoint of artifacts, manufacturing errors, and adjustment errors, the complex amplitude modulation region CAM has a shape extending radially around the optical axis AX as shown in FIGS. A rotationally symmetric shape having a spiral shape is desirable. In addition, it is desirable to have a shape that is longer in the radial direction than in the circumferential direction of the circle centered on the optical axis AX. For example, in the complex amplitude modulation region CAM of multiple annular zones as shown in FIG. 2C, the annular zones (complex amplitude modulation regions CAM) of a plurality of annular zones are easily arranged at equal intervals. Therefore, artifacts that emphasize specific frequency components are likely to occur. In addition, since the random amplitude complex amplitude modulation area CAM as shown in FIG. 2D has low periodicity, it is difficult to manufacture and adjust the direct light from the aperture IA so as to pass through the complex amplitude modulation area CAM. It is. On the other hand, in the rotationally symmetric complex amplitude modulation region CAM as shown in FIGS. 2A and 2B, the above disadvantages can be reduced. However, the desirable shape of the complex amplitude modulation region CAM varies depending on various conditions such as the object to be observed. For this reason, it is preferable to select an optimal complex amplitude modulation region CAM according to the purpose and the configuration of the apparatus.

  Next, a desirable size of the complex amplitude modulation region CAM will be described with reference to FIG. FIG. 3 is an explanatory diagram of a desirable size of the complex amplitude modulation region CAM. FIG. 3A shows a case where the complex amplitude modulation region CAM is relatively small, and FIG. 3B shows a case where the complex amplitude modulation region CAM is relatively small. Large cases are shown respectively.

  Here, a circle having a diameter R disposed on the modulation unit 105 is considered. The combined focal length of the lens 104a from the irradiated surface (sample 103) to the modulator 105 is f, the maximum wavelength of the light source 100 is λ (μm), and the numerical aperture on the irradiated surface side (sample 103 side) of the objective lens 104 is set. Let NA. Further, it is defined as Rn = 0.12 × λ / NA. At this time, when a circle having a diameter R represented by the following formula (1) is arranged at an arbitrary position in the complex amplitude modulation region CAM, the complex amplitude modulation region CAM is preferably a size that does not include this circle. . More preferably, the amplitude modulation area AM is also sized so as not to include this circle.

Rn = R / (NA × f) (1)
FIG. 3A shows the case where the circle C is not included in the complex amplitude modulation region CAM even if the circle C having the diameter R is arranged at an arbitrary position of the complex amplitude modulation region CAM. On the other hand, FIG. 3B shows a case where a circle C having a diameter R is included in the complex amplitude modulation region CAM when arranged at a position of the complex amplitude modulation region CAM.

  Here, Rn in Expression (1) is R normalized by the pupil diameter (NA × f) of the objective lens 104. Rn is determined by the first dark ring of the Airy disk. In the present embodiment, the assumed object diameter d of the sample 103 is 20 μm. For this reason, the diffraction angle θ of the object by the illumination light can be expressed as the following formula (2).

2sin θ = 2.44 × λ / d≈0.12 × λ (2)
At this time, the spread of the diffracted light on the pupil plane of the objective lens 104 is expressed as 2 sin θ × f. For this reason, the spread D of the diffracted light normalized by the pupil diameter NA × f of the objective lens 104 is expressed by the following equation (3). D represented by the formula (3) is equal to the aforementioned Rn.

D = (2 sin θ × f) / (NA × f)
= 0.12 × λ / NA (3)
The fact that the circle with the diameter R satisfying the equation (1) is not included in the complex amplitude modulation region CAM means that low-order diffracted light generated from a large object having an object diameter of about 20 μm does not enter the complex amplitude modulation region CAM. means. For this reason, satisfy | filling Formula (1), the diffracted light of various frequency components contributes to image formation, and the phase difference observation apparatus 10 can reproduce an image quantitatively.

  More preferably, it may be defined as Rn = 0.06 × λ / NA.

  The image obtained with the above-described configuration contains the diffracted light from the low order to the high order from the sample, and therefore has a quantitative intensity distribution I (x) and is expressed by the following formula (4). Can do.

I (x) = | U (x) -U ₀ (1-α · exp {iε})-n (U (x) -U ₀ ) (1-α · exp {iε})-(1-n) (U (x) −U ₀ ) (1-β) | ² (4)
Here, I (x) is the intensity distribution of the image when the intensity of the bright field image in the absence of an object is 1, U (x) is the complex amplitude transmittance distribution of the sample, and U ₀ is the average of U (x). Value. α is the amplitude transmittance of the complex amplitude modulation region CAM, β is the amplitude transmittance of the amplitude modulation region AM, ε is the phase modulation amount of the complex amplitude modulation region CAM, and n is the complex amplitude modulation region CAM occupying the pupil of the imaging optical system Ratio (0 <n <1), i is an imaginary unit. The first and second terms of the expression (4) are the same as those disclosed in Patent Document 1, and only the direct light represents the light component when the complex amplitude modulation is given by the complex amplitude modulation region CAM. . The third term represents a component of diffracted light that has been subjected to complex amplitude modulation by the complex amplitude modulation region CAM. Thus, according to the present invention, the diffracted light component that undergoes phase modulation by the complex amplitude modulation area CAM among the diffracted light from the sample, using the ratio n of the complex amplitude modulation area CAM occupying the pupil of the imaging optical system. Is calculated. The fourth term represents a component of diffracted light that has been subjected to amplitude modulation by the amplitude modulation area AM. Since I (x) is the intensity distribution of the image when the intensity of the bright field image in the absence of the object is 1, it is necessary to measure or estimate the intensity of the bright field image in the absence of the object. In this case, it can be estimated in consideration of the amplitude transmittance α of the complex amplitude modulation region CAM.

As can be seen from the equation (4), other than U (x) and U ₀ related to the sample are known. Therefore, U ₀ can be approximated to 1 if the sample is transparent, so U (x), that is, the phase distribution of the sample can be estimated from the intensity distribution I (x). That is, the phase difference amount distribution of the sample can be calculated based on the sample image information obtained by the image sensor 106 and the equation (4). In Expression (4), in addition to the first and second terms disclosed in Patent Document 1, as described above, the third term (diffracted light component that undergoes the same phase modulation as that of direct light) and the fourth term (amplitude different from that of direct light) The phase difference distribution of the sample is calculated based on the diffracted light component that is modulated). Also, if the sample has absorption, it is possible to obtain U (x) amplitude transmittance distribution and U ₀ by taking a bright field image in advance and calculating the square root of the image intensity. The phase distribution of U (x) can be estimated.

  Further, the intensity distribution I (x) _r of the actually obtained image is expected to change as shown in Expression (5) depending on the intensity modulation amount A and the offset amount B due to the influence of errors and the like.

I (x) _r = A · I (x) + B (5)
Therefore, when accurately estimating the phase distribution of the sample, use a plurality of images obtained under different conditions such as changing the phase modulation amount of the complex amplitude modulation region CAM or changing the wavelength when illuminating. Is desirable. Specifically, the phase difference of the sample 103 is determined from a plurality of pieces of image information obtained by changing at least one of the above-described amplitude transmittance α, amplitude transmittance β, phase modulation amount ε, and ratio n. It is desirable to calculate the quantity distribution. Further, the phase difference amount distribution of the sample 103 may be calculated from a plurality of pieces of image information obtained by changing the wavelength of the light source.

  In order to reduce the error when estimating the phase distribution of the sample using a white light source, the ratio n (0 <n <1) of the complex amplitude modulation region CAM occupying the pupil of the imaging optical system is as follows: It is preferable to satisfy Expression (6).

0.4 <n <0.6 (6)
This will be described with reference to FIG. FIG. 4 shows the phase distribution of the sample on the horizontal axis and the intensity of the image on the vertical axis using Equation (4). FIG. 4 shows how the image intensity becomes 400 nm and 800 nm when a phase difference amount of π / 2 is given to the phase modulation amount ε of the complex amplitude modulation region CAM at a wavelength of 600 nm. 4A and 4B show the case where the ratio n of the complex amplitude modulation area CAM occupying the pupil is 0.1 and 0.5, and the amplitude transmittance α of the complex amplitude modulation area CAM is not modulated. Without assuming a transparent phase object. As can be seen from FIG. 4A, when the ratio n of the complex amplitude modulation region CAM occupying the pupil is 0.1, the maximum intensity of the image and the peak position of the image intensity with respect to the phase distribution of the sample greatly change depending on the wavelength. ing. On the other hand, when the ratio n of the complex amplitude modulation region CAM occupying the pupil is 0.5 as shown in FIG. 4B, the maximum image intensity and the peak position of the image intensity with respect to the phase distribution of the sample do not change greatly depending on the wavelength. Therefore, when the phase distribution of the sample is assumed using a white light source, the ratio n of the complex amplitude modulation region CAM occupying the pupil is set to a value satisfying Equation (6), and n is set to a value close to 0.5. It is preferable.

  The main differences between the conventional general phase contrast microscope and dark field microscope and the configuration of the phase difference observation apparatus 10 of the present invention are the complex amplitude modulation area CAM, amplitude modulation area AM and illumination arranged in the objective lens. An aperture IA is arranged in the optical system. Therefore, the effect when these change is shown by simulation. The simulation condition is that the wavelength λ is 550 nm. The sample was a rectangular object whose phase increased linearly from 0 to 4π as shown in FIG. 5 in order to confirm that the image intensity quantitatively visualized the phase distribution. In this embodiment, the object is a completely transparent object.

  The phase difference observation apparatus 10 according to the first embodiment will be described in detail with reference to the simulation results at the time of NA 0.2 shown in FIGS. 6 and 7 below. In FIG. 6, the phase modulation amount ε of the complex amplitude modulation region CAM is π / 2, the amplitude transmittance α is 0.7, the amplitude transmittance β of the amplitude modulation region AM is 1, and the complex amplitude modulation region CAM and the opening IA are similar. It has a shape. FIG. 6 assumes a conventional general phase-contrast microscope, and the upper stage and the lower stage of FIG. 6A represent a complex amplitude modulation region CAM and an opening IA. 6B is a two-dimensional image of the simulation, FIG. 6C is a cross-sectional view of the object image of FIG. 6B cut in the vertical direction, and only a portion with the object is extracted, and the vertical axis is the image. Of strength. As can be seen from FIG. 6B, a change in image intensity is observed in the lateral direction of the object even though there is no phase distribution. Further, as can be seen from FIG. 6C, it can be seen that the phase difference amount of the object becomes discontinuous at 0 and 4π, and the image intensity has no periodicity. From these results, it is understood that the image intensity is not quantitative in a general phase contrast microscope.

  In FIG. 7, the phase modulation amount ε of the complex amplitude modulation region CAM is π / 2, the amplitude transmittance α is 0.7, the amplitude transmittance β of the amplitude modulation region AM is 1, and the complex amplitude modulation that occupies the pupil of the imaging optical system The ratio n of the area CAM is set to 0.5. 7A shows the complex amplitude modulation area CAM and the aperture IA, FIG. 7B shows the simulation two-dimensional image, and FIG. 7C shows the object image shown in FIG. 7B cut in the vertical direction. It is sectional drawing which extracted only the part with an object, and a vertical axis | shaft is the intensity | strength of an image. In FIG. 7C, the image intensity by simulation is indicated by a solid line, and the intensity estimated by the theoretical expression of Expression (4) is indicated by a dotted line.

  As shown in FIG. 7A, the complex amplitude modulation region CAM was set so as to extend radially at an angle of 5.625 ° with the optical axis AX as the center, and the angular pitch was 11.25 °. The length of the arc of the complex amplitude modulation region CAM extending radially is 0.087, and the right side of Equation (1) is 0.33. For this reason, the circle with the diameter R is not included in the complex amplitude modulation region CAM even if it is arranged at an arbitrary position in the complex amplitude modulation region CAM. As can be seen from FIG. 7B, unlike FIG. 6B, there is no change in image intensity in the horizontal direction of the image. Further, as can be seen from FIG. 7C, the intensity error between the solid line, which is the result of the simulation, and the dotted line estimated by the theoretical expression (4) is suppressed to 10% or less of the maximum intensity. From these results, it can be seen that in this embodiment, the phase distribution of the object is quantitatively visualized and the phase distribution of the object can be estimated correctly.

  The phase difference observation apparatus 10 according to the second embodiment will be described in detail with reference to the simulation result at NA 0.2 shown in FIG. Portions that are not particularly described are the same as those in the first embodiment.

  In FIG. 8, the phase modulation amount ε of the complex amplitude modulation region CAM is 0, the amplitude transmittance α is 0.25, the amplitude transmittance β of the amplitude modulation region AM is 1, and the complex amplitude modulation region CAM occupying the pupil of the imaging optical system The ratio n is 0.3. 8A shows the complex amplitude modulation area CAM and the aperture IA, FIG. 8B shows the simulation two-dimensional image, and FIG. 8C shows the object image shown in FIG. 8B cut in the vertical direction. It is sectional drawing which extracted only the part with an object, and a vertical axis | shaft is the intensity | strength of an image. In FIG. 8C, the image intensity by simulation is indicated by a solid line, and the intensity estimated by the theoretical expression of Expression (4) is indicated by a dotted line.

  The diameter of each dot of the complex amplitude modulation area CAM, which is a random dot, is 0.05, and the right side of Equation (1) is 0.33. For this reason, the circle with the diameter R is not included in the complex amplitude modulation region CAM even if it is arranged at an arbitrary position in the complex amplitude modulation region CAM. As can be seen from FIG. 8B, unlike FIG. 6B, there is no change in image intensity in the horizontal direction of the image. Further, as can be seen from FIG. 8C, the intensity error between the solid line, which is the result of the simulation, and the dotted line estimated by the theoretical expression of Expression (4) is suppressed to 10% or less of the maximum intensity. From these results, it can be seen that in this embodiment, the phase distribution of the object is quantitatively visualized and the phase distribution of the object can be estimated correctly.

  The phase difference observation apparatus 10 according to the third embodiment will be described in detail with reference to the simulation result at NA 0.2 shown in FIG. Portions that are not particularly described are the same as those in the first embodiment.

  In FIG. 9, the phase modulation amount ε of the complex amplitude modulation region CAM is 0, the amplitude transmittance α is 1, the amplitude transmittance β of the amplitude modulation region AM is 0, and the ratio of the complex amplitude modulation region CAM in the pupil of the imaging optical system n is set to 0.5. 9A shows the complex amplitude modulation region CAM and the aperture IA, FIG. 9B shows the simulation two-dimensional image, and FIG. 9C shows the object image shown in FIG. 9B cut in the vertical direction. It is sectional drawing which extracted only the part with an object, and a vertical axis | shaft is the intensity | strength of an image. In FIG. 9C, the image intensity by simulation is indicated by a solid line, and the intensity estimated by the theoretical expression of Expression (4) is indicated by a dotted line.

  Each annular width of the complex amplitude modulation region CAM which is a multiple annular zone is 0.05, and the right side of Equation (1) is 0.33. For this reason, the circle with the diameter R is not included in the complex amplitude modulation region CAM even if it is arranged at an arbitrary position in the complex amplitude modulation region CAM. As can be seen from FIG. 9B, unlike FIG. 6B, there is no change in image intensity in the horizontal direction of the image. Further, as can be seen from FIG. 9C, the intensity error between the solid line, which is the result of the simulation, and the dotted line estimated by the theoretical expression of Expression (4) is suppressed to 10% or less of the maximum intensity. From these results, it can be seen that in this embodiment, the phase distribution of the object is quantitatively visualized and the phase distribution of the object can be estimated correctly.

  The phase difference observation apparatus 10 according to the fourth embodiment will be described in detail with reference to a simulation result at NA 0.7 shown in FIG. Parts not specifically mentioned are the same as those in the first embodiment.

  In FIG. 10, the phase modulation amount ε of the complex amplitude modulation region CAM is 3π / 2, the amplitude transmittance α is 1, the amplitude transmittance β of the amplitude modulation region AM is 1, and the complex amplitude modulation region CAM occupying the pupil of the imaging optical system The ratio n is set to 0.5. 10A shows the complex amplitude modulation region CAM and the aperture IA, FIG. 10B shows the simulation two-dimensional image, and FIG. 10C shows the object image shown in FIG. 10B cut in the vertical direction. It is sectional drawing which extracted only the part with an object, and a vertical axis | shaft is the intensity | strength of an image. In FIG. 10C, the image intensity by simulation is indicated by a solid line, and the intensity estimated by the theoretical expression of Expression (4) is indicated by a dotted line.

  As shown in FIG. 10A, the complex amplitude modulation region CAM was set so as to extend radially at an angle of 2.8125 ° around the optical axis AX and to have an angular pitch of 5.625 °. The length of the arc of the complex amplitude modulation region CAM extending radially is 0.044, and the right side of Equation (1) is 0.094. For this reason, the circle with the diameter R is not included in the complex amplitude modulation region CAM even if it is arranged at an arbitrary position in the complex amplitude modulation region CAM. As can be seen from FIG. 10B, unlike FIG. 6B, there is no change in image intensity in the horizontal direction of the image. Further, as can be seen from FIG. 10C, the intensity error between the solid line, which is the result of the simulation, and the dotted line estimated by the theoretical expression (4) is suppressed to 10% or less of the maximum intensity. From these results, it can be seen that in this embodiment, the phase distribution of the object is quantitatively visualized and the phase distribution of the object can be estimated correctly.

  ADVANTAGE OF THE INVENTION According to this invention, the phase difference observation apparatus which visualizes a phase distribution quantitatively with low calculation cost from the acquired image can be provided, without changing the structure of the conventional phase-contrast microscope and a dark field microscope significantly. That is, according to the present invention, it is possible to provide a phase difference observation apparatus that is advantageous for visualizing the phase distribution of a sample.

  The preferred embodiments of the present invention have been described above, but the present invention is not limited to these embodiments, and various modifications and changes can be made within the scope of the gist. For example, although the transmission illumination is used in this embodiment, it may be configured as epi-illumination.

  For example, when a software program that implements the functions of the above-described embodiments is supplied from a recording medium directly to a system or apparatus having a computer that can execute the program using wired / wireless communication, and the program is executed Are also included in the present invention.

  Accordingly, the program code itself supplied and installed in the computer in order to implement the functional processing of the present invention by the computer also realizes the present invention. That is, the present invention includes a computer program itself in which a procedure for realizing the functional processing of the present invention is described.

  In this case, the program may be in any form as long as it has a program function, such as an object code, a program executed by an interpreter, or script data supplied to the OS. The recording medium for supplying the program may be, for example, a magnetic recording medium such as a hard disk or a magnetic tape, an optical / magneto-optical storage medium, or a nonvolatile semiconductor memory.

  As a program supply method, a computer program that forms the present invention is stored in a server on a computer network, and a connected client computer downloads and programs the computer program.

  The present invention can be suitably used for a phase contrast microscope or a dark field microscope for observing a biological sample or a biological tissue.

DESCRIPTION OF SYMBOLS 10 Phase difference observation apparatus 104 Objective lens 105 Modulation part 106 Image sensor 107 Image processing system

Claims (16)

An imaging optical system for forming an image of the sample;
A modulation unit provided in the imaging optical system and having a first modulation region for modulating incident light;
Imaging means for imaging the sample via the imaging optical system;
Calculation means for calculating a phase difference amount distribution of the sample based on image information obtained by the imaging means and a diffracted light component modulated by the first modulation region among diffracted light from the sample; An observation apparatus comprising:   The observation apparatus according to claim 1, wherein the calculation unit calculates the diffracted light component using a ratio of the first modulation region to a pupil of the imaging optical system. The calculating means sets the intensity distribution of the image when the intensity of the bright field image in the absence of an object is 1, I (x), the complex amplitude transmittance distribution of the sample is U (x), and the U (x) U ₀ , the amplitude transmittance of the first modulation region is α, the amplitude transmittance of the second modulation region different from the first modulation region in the modulator is β, and the phase modulation of the first modulation region When the amount is ε, the ratio of the first modulation region in the pupil of the imaging optical system is n (0 <n <1), and the imaginary unit is i,
I (x) = | U (x) -U ₀ (1-α · exp {iε})-n (U (x) -U ₀ ) (1-α · exp {iε})-(1-n) (U (x) -U ₀ ) (1-β) | ²
The observation apparatus according to claim 1, wherein a phase difference amount distribution of the sample is calculated based on the following formula.   The calculating means includes an amplitude transmittance α of the first modulation region, an amplitude transmittance β of the second modulation region, a phase modulation amount ε of the first modulation region, and the first occupying the pupil of the imaging optical system. The phase difference amount distribution of the sample is calculated based on a plurality of pieces of image information obtained by imaging at least one of the modulation area ratios n (0 <n <1). The observation apparatus according to claim 3. An illumination optical system that illuminates the sample with light from a light source;
The illumination optical system has an aperture stop provided with an aperture,
The modulation unit is disposed at a position conjugate with the opening, and has a second modulation region different from the first modulation region,
The first modulation region is arranged so that direct light from the light source that has passed through the opening is incident;
The observation apparatus according to claim 1, wherein the second modulation region is arranged so that the direct light is not incident thereon. The composite focal length from the sample to the modulation unit is f, the numerical aperture on the sample side of the imaging optical system is NA, the maximum wavelength of the light source is λ (μm), Rn = 0.12 × λ / NA , And when
Rn = R / (NA × f)
6. When a circle having a diameter R that satisfies the following formula is arranged at an arbitrary position of the modulation section, the first modulation area or the second modulation area is a size that does not include the circle. The observation apparatus described in 1.   7. The observation according to claim 5, wherein the calculation unit calculates a phase difference amount distribution of the sample based on a plurality of pieces of image information obtained by imaging while changing the wavelength of the light source. apparatus.   The first modulation region has a shape that is rotationally symmetric about an optical axis, and has a shape that is longer in a radial direction than a circumferential direction of a circle around the optical axis. The observation apparatus according to any one of the above.   The observation apparatus according to claim 8, wherein the first modulation region has a shape extending radially around the optical axis or a spiral shape. The ratio n (0 <n <1) of the first modulation area in the pupil of the imaging optical system is
0.4 <n <0.6
The observation apparatus according to claim 1, wherein the following expression is satisfied.   Image information of a sample obtained by imaging through a modulation unit having a first modulation region provided in the imaging optical system, and a diffracted light component modulated by the first modulation region of the diffracted light from the sample, And a calculation step of calculating a phase difference amount distribution of the sample based on the calculation method.   The calculation method according to claim 11, wherein the calculating step calculates the diffracted light component using a ratio of the first modulation region occupying a pupil of the imaging optical system. In the calculation step, the intensity distribution of the image when the intensity of the bright field image in the absence of an object is 1 is I (x), the complex amplitude transmittance distribution of the sample is U (x), and the U (x) U ₀ , the amplitude transmittance of the first modulation region is α, the amplitude transmittance of the second modulation region different from the first modulation region in the modulator is β, and the phase modulation of the first modulation region When the amount is ε, the ratio of the first modulation region in the pupil of the imaging optical system is n (0 <n <1), and the imaginary unit is i,
I (x) = | U (x) -U ₀ (1-α · exp {iε})-n (U (x) -U ₀ ) (1-α · exp {iε})-(1-n) (U (x) -U ₀ ) (1-β) | ²
The calculation method according to claim 11 or 12, wherein a phase difference amount distribution of the sample is calculated based on the following formula.   Image information of a sample obtained by imaging through a modulation unit having a first modulation region provided in the imaging optical system, and a diffracted light component modulated by the first modulation region of the diffracted light from the sample, And a calculation means for calculating a phase difference distribution of the sample based on the above.   15. The arithmetic device according to claim 14, wherein the calculation unit calculates the diffracted light component using a ratio of the first modulation region occupying a pupil of the imaging optical system. The calculating means sets the intensity distribution of the image when the intensity of the bright field image in the absence of an object is 1, I (x), the complex amplitude transmittance distribution of the sample is U (x), and the U (x) U ₀ , the amplitude transmittance of the first modulation region is α, the amplitude transmittance of the second modulation region different from the first modulation region in the modulator is β, and the phase modulation of the first modulation region When the amount is ε, the ratio of the first modulation region in the pupil of the imaging optical system is n (0 <n <1), and the imaginary unit is i,
I (x) = | U (x) -U ₀ (1-α · exp {iε})-n (U (x) -U ₀ ) (1-α · exp {iε})-(1-n) (U (x) -U ₀ ) (1-β) | ²
The arithmetic unit according to claim 14 or 15, wherein a phase difference amount distribution of the sample is calculated based on the following formula.