JP5091466B2 - Observation device - Google Patents

Description

  The present invention relates to an observation apparatus for observing a transparent object.

For observation of transparent samples such as cells in a culture vessel, phase difference observation that can visualize the refractive index distribution is suitable (Non-Patent Documents 1 and 2, etc.). A transparent sample that cannot be observed with the naked eye requires a microscope or some optical system even if only a rough state of the entire sample is confirmed.
Kei Komatsu, “Basics and Applications of Optical Microscope (3)”, Applied Physics, Vol. 60, No. 10, 1991, p1032-p1034 Kei Komatsu, “Basics and Applications of Optical Microscope (4)”, Applied Physics, Vol. 60, No. 11, 1991, p1136-p1138

However, there is no phase-contrast microscope that has a wide field of view so that the entire area of the culture vessel can be observed all at once. To achieve this, an extra large phase difference imaging optical system and an extra large ring illumination optical system are required. So it is very expensive.
In addition, if only a rough state of the entire sample is confirmed, dark field observation may be applied instead of phase difference observation. In the dark field observation, weak diffracted light (scattered light) generated at the step of the refractive index in the sample is captured, and an observation image is formed only by the diffracted light.

However, when the dark field microscope has a wide field of view, the illumination angle becomes large, and an observation image can be formed only with very weak diffracted light emitted from the sample at a large angle, so that the brightness is remarkably insufficient. In addition, if the illumination angle is large, strong light generated at the edge or bottom of the culture vessel may enter the imaging optical system, which may obstruct observation.
Therefore, an object of the present invention is to provide an observation apparatus suitable for observing a transparent object with a wide field of view.

The observation apparatus of the present invention is arranged with a surface light source formed by repeatedly arranging a bright part and a dark part on a surface away from the observation surface of the object to be observed, and an illuminating means for illuminating the observation surface from the arrangement surface , An imaging optical system that captures, at the pupil position, diffracted light that has passed through the partial area facing the dark part of the observation surface illuminated by the illuminating means, and forms a dark field image of the partial area with the diffracted light. And an image pickup means for picking up the dark field image formed by the image forming optical system, wherein the surface light source is disposed on the image forming surface of the image forming optical system. The distance from the surface light source to the observation surface is out of the depth of field, and the brightness distribution in the pitch direction of the bright and dark portions of the image of the surface light source formed on the imaging surface of the imaging means is sinusoidal. It is set to a value such that
Note that the above distance d, and the pitch P of brightness of the surface light source, compared object-side numerical aperture NA of the imaging optical system, P / d ≦ √ [2 {1-0.02 / (NA 2) }] Is desirable.

Moreover, said distance d, and the pitch P of brightness of the surface light source, 'to, P / d ≦ 2 · NA' image-side numerical aperture NA of the imaging optical system preferably satisfies the equation.
Moreover, said distance d, and the pitch P of brightness of the surface light source, the imaging optical system object-side numerical aperture NA of the wavelength lambda, the the image-side principal plane of the imaging optical system imaging the surface light source It is desirable to satisfy the formula of λ / (2 · a · NA) ≦ P / d with respect to the distance a to the means.

The illumination means may include a surface light source that emits light with substantially uniform intensity, and a mask that is disposed on the surface light source and in which an opening portion and a light shielding portion are repeatedly disposed.
The illumination means may be provided with a liquid crystal spatial modulation element.
The observation apparatus of the present invention may further include image processing means for removing a component of the image of the surface light source from the image of the observation surface acquired by the imaging means.

The observation apparatus of the present invention may further include means for changing the light / dark phase of the surface light source.
Moreover, the observation apparatus of the present invention may further include means for changing a light / dark pitch of the surface light source.
The observation apparatus of the present invention may further include means for changing a light / dark duty ratio of the surface light source.
Another observation apparatus according to the present invention forms an image of an illumination unit that illuminates an observation object with a surface light source in which a bright part and a dark part are arranged, and light transmitted through the observation object illuminated by the illumination unit. An imaging optical system that performs imaging, and an imaging unit that captures an image of the observation object formed by the imaging optical system, wherein the surface of the surface light source deviates from the depth of field of the imaging optical system. The image of the surface light source formed on the imaging surface of the imaging unit by the imaging optical system is blurred to such an extent that the presence of the bright part and the dark part can be recognized.

  ADVANTAGE OF THE INVENTION According to this invention, the observation apparatus suitable for observing a transparent to-be-observed object with a wide visual field is implement | achieved.

[First Embodiment]
Hereinafter, the first embodiment will be described. This embodiment is an embodiment of an observation apparatus.
FIG. 1 is an overall configuration diagram of the observation apparatus. As shown in FIG. 1, this observation apparatus includes a transmissive liquid crystal panel 14 with a backlight, a stage (sample stage) 13, an imaging optical system 12, an image sensor 11, a transmissive liquid crystal panel controller 15, a computer 16, A display 17 or the like is provided.

A transparent object 10 is placed on the stage 13. The observation object 10 is, for example, a transparent culture container (for example, a petri dish having a diameter of 35 mm) in which unstained cells are stored.
The transmissive liquid crystal panel 14 illuminates substantially the entire area of the observation object 10. The portion of the stage 13 on which the object to be observed 10 is placed is made of a transparent member such as glass or is hollow so that the illumination light is not blocked.

  The field of view of the imaging optical system 12 is sufficiently large, and light emitted from substantially the entire area of the object to be observed 10 is captured by the imaging optical system 12 and forms an image on the imaging surface of the imaging element 11. The image sensor 11 acquires an image (luminance distribution) on the imaging surface in response to an instruction from the computer 16. The image is captured by the computer 16 and displayed on the display 17 after image processing.

In addition, the computer 16 can preserve | save the image before image processing, and the image after image processing as needed. In addition, an image before image processing can be displayed on the display 17 as necessary.
Here, in response to an instruction from the computer 16, the controller 15 displays a pattern formed by repeatedly arranging a bright part and a dark part on the transmissive liquid crystal panel 14. Here, this pattern is a stripe pattern. At this time, a striped surface light source is formed on the transmissive liquid crystal panel 14.

The pitch P of the surface light source is sufficiently larger than the size (10 to 15 μm) of the phase object (cell) to be observed, and the distance d from the surface light source to the object to be observed 10 is the surface light source. Is appropriately distant from the depth of field of the imaging optical system 12. The relationship between the pitch P and the interval d is set so as to satisfy the following three conditions.
(Condition 1) When viewed from the partial area on the object 10 facing the dark part of the surface light source, the angle between the two bright parts on both sides of the dark part is sufficiently small.
(Condition 2)... Both the brightness and darkness of the surface light source are surely present in the field of the imaging optical system 12.
(Condition 3)... The presence or absence of brightness of the surface light source can be reliably recognized on the image sensor 11.

Details of these conditions 1, 2, and 3 will be described later.
In particular, here, it is assumed that several tens of bright and dark pitches of the surface light source exist in the field of the imaging optical system 12, and the ratio (duty ratio) between the width of the bright portion and the width of the dark portion of the surface light source is 50%. To do. Here, it is assumed that the surface light source image on the image sensor 11 is moderately blurred, and the luminance distribution curve in the pitch direction of the surface light source image draws a sine wave. That is, the illuminance of the surface light source image is substantially uniform on the observation surface 10a. Note that either a binary pattern or a gray scale pattern may be adopted as the display pattern on the transmissive liquid crystal panel 14.

FIG. 2 is a conceptual diagram illustrating the operation of the optical system portion of the present observation apparatus. FIG. 2 shows the concept of the optical system portion of the present observation apparatus.
First, description will be made by paying attention to a certain dark portion 14A on the surface light source and a partial region 10A on the object 10 to be observed facing the dark portion 14A.
Lights LB and LC from two bright portions 14B and 14C adjacent to the dark portion 14A are incident on the partial region 10A. Light does not enter from the dark portion 14A. Therefore, the partial area 10A is illuminated obliquely with the light LB and LC.

  In the partial region 10A, a part of the incident light LB and LC is transmitted without being diffracted, but the other part of the light LB and LC is affected by the step of the refractive index in the partial region 10A. Diffracted (or scattered). The non-diffracted lights LB and LC transmitted through the partial area 10A as they are do not enter the pupil to the imaging optical system 12, but some diffracted lights (or scattered lights) Lb and Lc generated in the partial area 10A are The light enters the pupil of the imaging optical system 12.

At this time, in the region 11A on the image sensor 11 conjugated with the partial region 10A, an image of the dark part 14A and an image of the outline of the phase object existing in the partial region 10A (hereinafter referred to as “observed image”). Are formed to overlap. That is, a dark field image of the partial region 10A is formed.
Here, when viewed from the partial region 10A, the angle between the two bright portions 14B and 14C is sufficiently small. Therefore, the illumination angle of the region 10A with the light LB and LC is also sufficiently small. At this time, the observation image of the partial region 10A is generated by the high-intensity diffracted lights Lb and Lc emitted at a small angle. Therefore, the brightness of the observation image in the partial region 10A is sufficiently high.

In addition, the above applies to each region of the object to be observed 10 facing each dark part. Therefore, the image I ₁ acquired by the image sensor 11 is obtained by superimposing an observation image on a striped dark background, as shown in FIG. In FIG. 3, the boundary between the bright and dark portions of the surface light source image is sharply shown, but is actually blurred.
By displaying the image I ₁ computer 16 on the display 17, the state of the observed object 10 users can be easily observed.

However, what appears in this image I ₁ is only an observation image of a substantially half region of the object to be observed 10 (only an observation image of a region facing the dark part). Moreover, since the background of the observed image has a sinusoidal luminance distribution rather than a complete dark portion, the contrast of the observed image is low.
Therefore, the computer 16 of the present observation apparatus executes the following procedures so that substantially the entire area of the object to be observed 10 can be observed with high contrast.
(Step 1) ... displays a transmissive liquid crystal panel 14 striped surface illuminant on (duty ratio 50%), to obtain the image I ₁ as shown in FIG. 3 in that state.
(Step 2) ... the dark phase of the surface light source is shifted by [pi, acquires an image I ₂ as shown in FIG. 4 in this state. In this image I ₂ , an observation image of an area different from that of the image I ₁ appears. The concept of the luminance distribution in the light and dark pitch directions of these images I ₁ and I ₂ is shown in FIG.
(Procedure 3) As shown in FIGS. 5A to 5B, the images I ₁ and I ₂ are individually subjected to spatial Fourier transform, and the data D ₁ representing the image I ₁ for each spatial frequency component, and the image I ₂ the obtaining and data D ₂ representing each spatial frequency component. In these data D ₁ and D ₂ , a large peak on the low frequency side is a component of the surface light source image, and a plurality of small peaks on the high frequency side are components of the observation image.
(Step 4) ... as shown in FIG. 5 (b) → (c), to remove the components of the surface light source images from each of the data D _1, D _2, data D ₁ ', D _2' acquires.
(Procedure 5)... As shown in FIGS. 5C to 5D, data D ₁ ′ and D ₂ ′ are individually subjected to inverse Fourier transform to obtain images I ₁ ′ and I ₂ ′. In these images I ₁ and I ₂ , the surface light source image is removed, and only the observation image appears.
(Procedure 6)... As shown in FIGS. 5D to 5E, the images I ₁ ′ and I ₂ ′ are combined to obtain one image I. This image I is displayed on the display 17.

In this image I, an observation image of almost the entire area of the object to be observed 10 appears, and the background of the observation image is completely dark. Therefore, the user can perform dark field observation with high contrast over substantially the entire area of the object to be observed 10.
[Second Embodiment]
The second embodiment will be described below. Here, only differences from the first embodiment will be described. The difference is in the processing procedure by the computer 16. The procedure of this embodiment is as follows.
(Step 1) ... displays a transmissive liquid crystal panel 14 striped surface illuminant on (duty ratio 50%), to obtain the image I ₁ as shown in FIG. 6 (a) in that state.
(Procedure 2)... The phase of the surface light source is shifted by 2π / 3, and an image I ₂ as shown in FIG. 6B is acquired in this state.
(Procedure 3)... The phase of light and darkness of the surface light source is further shifted by 2π / 3, and an image I ₃ as shown in FIG.
(Procedure 4)... The images I ₁ , I ₂ , and I ₃ are applied to the following arithmetic expression (1) for each pixel to obtain one image I as shown in FIG.

I = √ [(I ₁ −I ₂ ) ² + (I ₂ −I ₃ ) ² + (I ₃ −I ₁ ) ² ] (1)
In this image I, an observation image of substantially the entire area of the object to be observed 10 appears, and the background of the observation image has a uniform brightness. In order to darken the background, the computer 16 removes a component having zero spatial frequency (0th order component) from the image I and displays the image after the removal on the display 17.

In the image after removal, an observation image of substantially the entire area of the object to be observed 10 appears, and the background of the observation image is completely dark. Therefore, the user can perform dark field observation with high contrast over substantially the entire area of the object to be observed 10.
In the present embodiment, a simple arithmetic expression (1 ′) can be used instead of the arithmetic expression (1).

I = I ₁ + I ₂ + I ₃ (1 ′)
[Conditions 1, 2, and 3]
Details of the conditions 1, 2, and 3 will be described below.
FIG. 7 is a diagram illustrating conditions 1, 2, and 3. In FIG. 7, reference numeral 11 a is an imaging surface of the imaging device 11, reference numeral 10 a is a focal plane (= observation plane) of the imaging optical system 12, and reference numeral 14 a is a surface light source formation surface (= light source plane). It is.

(Condition 1)
First, an angle between the dark portion 14A and the bright portion 14C when viewed from the observation surface 10a is set to θ. This angle θ corresponds to the incident angle of the two lights LC and LB that illuminate the observation surface 10a.
At this time, the diffraction angles of the diffracted lights Lc and Lb that can enter the pupil of the imaging optical system 12 from the observation surface 10a are angles ξ that simultaneously satisfy the following expressions (2), (3), and (4). is there.

cosξ = cosθ · cosρ + sinθ · sinρ · cosφ (2)
0 ≦ ρ ≦ NA (3)
0 ≦ φ ≦ 2π (4)
However, NA is the object-side numerical aperture of the imaging optical system 12, and can be approximated by assuming that it is sufficiently small.

From these formulas (2), (3), and (4), the light quantity ratio R of the diffracted lights Lc and Lb based on the light quantity of the lights LC and LB that illuminate the observation surface 10a is expressed by the formula (5). The
R = (1/2) · ∫cosξ · dρ · dφ
= (1/2) · NA ² · cos θ (5)
If this light quantity ratio R is sufficiently high, the brightness of the observation image is sufficiently secured. Therefore, the light quantity ratio R needs to satisfy at least the following formula (6).

R ≧ 0.01 (6)
Substituting equation (5) into equation (6) yields equation (7).
(1/2) · NA ² · cos θ ≧ 0.01 (7)
Here, the angle θ is given by the following equation (8) by the distance d from the light source surface 14a to the observation surface 10a and the light / dark pitch P of the surface light source. However, the approximation was made assuming that θ is small.

θ = (P / 2) / d (8)
Substituting this equation (8) into equation (7) yields equation (9). However, theta is approximated regarded as sufficiently small (cos [theta] ≒ using 1-θ ^2/2 of the approximate expression).
P / d ≦ √ [2 {1-0.02 / (NA ² )}] (9)
This expression (9) is a conditional expression corresponding to condition 1.

(Condition 2)
First, the width Z of the region of the light source surface 14a that falls within the field of view of the imaging optical system 12 is expressed by Expression (10), where NA ′ is the image-side numerical aperture of the imaging optical system 12.
Z = 2 · NA ′ × d (10)
In addition, in order for light and darkness of one pitch or more to exist in the region within the width Z on the light source surface 14a, the expression (11) needs to be satisfied.

P ≦ Z (11)
Substituting equation (10) into equation (11) yields equation (12).
P / d ≦ 2NA ′ (12)
This expression (12) is a conditional expression corresponding to condition 2.
(Condition 3)
First, in order for the light and darkness on the light source surface 14a to be resolved on the imaging surface 11a, the expression (13) needs to be satisfied with respect to the magnification M of the imaging optical system 12 and the light source wavelength λ.

P · M ≧ λ / (2 · NA) (13)
The magnification M of the imaging optical system 12 is set such that the distance from the image side main surface of the imaging optical system 12 to the imaging surface 11a is a, and the distance from the image side main surface of the imaging optical system 12 to the observation surface 10a. When the interval is b, it is expressed by the following formula (14).
M = a / (b + d) (14)
Here, since d >> b can be considered, Expression (14) is rewritten to Expression (15).

M = a / d (15)
Substituting this equation (15) into equation (13) yields equation (16).
λ / (2 · a · NA) ≦ P / d (16)
This expression (16) is a conditional expression corresponding to condition 3.
[Modification of each embodiment]
In each embodiment, a transmissive liquid crystal panel with a backlight and a controller are used to generate a surface light source, but other devices may be used as long as the same surface light source can be generated. .

  For example, a large number of small light sources such as LEDs are prepared, and they are densely arranged in an array. Thus, a surface light source that emits light with substantially uniform intensity is formed. Then, a mask member having a striped opening is disposed on the surface light source. Thereby, a striped surface light source is formed. Incidentally, in order to shift the light / dark phase of the surface light source, the mask member may be shifted in the light / dark pitch direction. Further, instead of shifting the mask member, a plurality of mask members having different opening patterns may be prepared and switched.

In each embodiment, the brightness pitch of the surface light source is not changed, but the pitch may be changed according to the size of the phase object (cell) to be observed. Then, phase objects (cells) of various sizes can be favorably observed.
In each embodiment, the light / dark duty ratio of the surface light source is not changed. However, the duty ratio may be changed according to the refractive index of the phase object (cell) to be observed. For example, since the intensity of diffracted light increases when the refractive index of the phase object (cell) is sufficiently high, the dark portion ratio is increased (70%, 80%, etc.), and the number of image acquisitions is reduced accordingly. Good. This is because if the ratio of the dark part is large, an observation image of a wider area appears on the same image. On the other hand, when the refractive index of the phase object (cell) is low, the ratio of the dark part may be lowered (30% or the like), and the number of image acquisitions may be increased accordingly.

  In each embodiment, the light / dark pattern of the surface light source is a stripe pattern, but other patterns in which bright and dark portions appear repeatedly may be employed. For example, a checkered pattern as shown in FIG. 8 can be adopted. Also, a concentric pattern as shown in FIG. 9 can be adopted. In either case, when shifting the phase of light and dark, the pattern may be changed in the direction in which light and dark are reversed (in the case of a concentric circular pattern, the radial direction).

  Incidentally, it is preferable to employ a stripe pattern (FIG. 1) or a checkered pattern (FIG. 8) because the contents of processing for shifting the phase and image processing are simplified.

NA ≧ 0.14286,
P = 6mm,
d = 30 mm,
These numerical values satisfy Condition 1 (Formula (9)).

NA ′ ≧ 0.1,
P = 6mm,
d = 30 mm,
These numerical values satisfy Condition 2 (Formula (12)).

NA ≧ 0.003
P = 6mm,
d = 30 mm,
a = 5 mm,
λ = 550 nm = 550 × 10 ⁻⁴ mm,
These numerical values satisfy Condition 3 (Formula (16)).

NA ≧ 0.14286,
P = 6mm,
d = 30 mm,
NA ′ ≧ 0.1,
a = 5 mm,
λ = 550 nm = 550 × 10 ⁻⁴ mm
These numerical values satisfy all of Condition 1 (Expression (9)), Condition 2 (Expression (12)), and Condition 3 (Expression (16)).

It is a whole block diagram of an observation apparatus. It is a conceptual diagram explaining the effect | action of the optical system part of an observation apparatus. It is a diagram illustrating an image I _1. Is a diagram showing an image I _2. It is a figure explaining the procedure of the image processing by the computer 16 of 1st Embodiment. It is a figure explaining the procedure of the image processing by the computer 16 of 2nd Embodiment. It is a figure explaining conditions 1, 2, and 3. FIG. It is a figure which shows the modification of the light-and-dark pattern of a surface light source. It is a figure which shows another modification of the light-dark pattern of a surface light source.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Imaging device, 12 ... Imaging optical system, 13 ... Stage, 14 ... Transmission-type liquid crystal panel, 15 ... Controller, 16 ... Computer, 17 ... Display

Claims (10)

A surface light source formed by repeatedly arranging a bright part and a dark part is disposed on a surface away from the observation surface of the object to be observed, and illumination means for illuminating the observation surface from the arrangement surface ;
An imaging optical system that captures, at the pupil position, diffracted light that has passed through the partial area facing the dark part of the observation surface illuminated by the illuminating means, and forms a dark field image of the partial area with the diffracted light. When,
An imaging unit that is disposed on an imaging surface of the imaging optical system and that captures the dark field image formed by the imaging optical system;
An arrangement surface of the surface light source is out of the depth of field of the imaging optical system, and an interval from the surface light source to the observation surface is an image of the surface light source formed on the imaging surface of the imaging unit. An observation device characterized in that the brightness distribution in the pitch direction of the bright part and the dark part is set to a value that is sinusoidal . The observation apparatus according to claim 1,
The distance d and the light / dark pitch P of the surface light source are defined with respect to the object-side numerical aperture NA of the imaging optical system.
P / d ≦ √ [2 {1-0.02 / (NA ² )}]
An observation apparatus characterized by satisfying the formula: In the observation device according to claim 1 or 2,
The distance d and the light / dark pitch P of the surface light source are relative to the image-side numerical aperture NA ′ of the imaging optical system.
P / d ≦ 2 · NA '
An observation apparatus characterized by satisfying the formula: In the observation apparatus as described in any one of Claims 1-3,
The distance d and the light / dark pitch P of the surface light source include the object-side numerical aperture NA of the imaging optical system, the wavelength λ of the surface light source, the image-side main surface of the imaging optical system, and the imaging means. For the interval a of
λ / (2 · a · NA) ≦ P / d
An observation apparatus characterized by satisfying the formula: In the observation apparatus according to any one of claims 1 to 4,
The illumination means includes
A surface light source that emits light with substantially uniform intensity;
An observation apparatus, comprising: a mask that is disposed on the surface light source and in which an opening portion and a light shielding portion are repeatedly disposed. In the observation apparatus according to any one of claims 1 to 4,
The illumination means includes
An observation apparatus comprising a liquid crystal spatial modulation element. In the observation apparatus as described in any one of Claims 1-6,
An observation apparatus further comprising image processing means for removing a component of the image of the surface light source from the image of the observation surface acquired by the imaging means. In the observation apparatus according to any one of claims 1 to 7,
An observation apparatus further comprising means for changing a light / dark phase of the surface light source. In the observation apparatus according to any one of claims 1 to 8,
An observation apparatus further comprising means for changing a light / dark pitch of the surface light source. In the observation apparatus according to any one of claims 1 to 9,
An observation apparatus, further comprising means for changing a light / dark duty ratio of the surface light source.

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