JP2003015049A - Lattice illumination microscope and observation method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an image with good picture quality even when a pixel pitch is not sufficiently small to that of a lattice pattern illuminated on a specimen. SOLUTION: An lattice illumination microscope is provided with a lighting source to emit illumination light with one dimensional lattice pattern from one dimensional lattice 3, a specimen placing plate 15 to place the specimen 16, an imaging optical system to form the image of the specimen in response to observation light emitted from the specimen on the specimen placing plate in response to the illumination light with the lattice pattern, and a CCD camera 8 to photograph the image of the specimen formed by the imaging optical system. The microscope has a direction regulating device 12 to regulate the rotational position of the direction of the lattice pattern in the image of the specimen photographed by the CCD camera 8 and the array direction of the pixel in an imaging device.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lattice illumination microscope for illuminating a specimen in a lattice pattern and a method for observing a specimen using the lattice illumination microscope.

[0002]

2. Description of the Related Art An ordinary optical microscope is not suitable for observing a three-dimensional structure (three-dimensional structure) of a specimen, but this will be briefly described. An ordinary optical microscope can only obtain an image that is the same as the image at the time of focusing only by defocusing if there is no fine structure in the sample when defocusing. It should be noted that if the sample has a fine structure, the appearance of the fine structure may change depending on the defocus, but when the focus is blurred, the integrated intensity of the light amount of the entire image hardly changes, so Even if there was a change in the appearance of the fine structure due to the shift, it was difficult to observe the change due to the background light. As can be seen from this, it is difficult to obtain information change in the optical axis direction of the optical system in the sample with a normal microscope, and therefore it is not suitable for observing the three-dimensional structure (three-dimensional structure) of the sample. .

On the other hand, there is a confocal microscope as a microscope capable of observing the three-dimensional structure of a sample. The confocal microscope illuminates the sample in a point shape, and again forms an image of the light scattered at the illuminating point and captures it with a point-like photodetector. To be able to get. Therefore, by scanning the illumination point two-dimensionally on the surface of the sample, not only the two-dimensional information of the sample that can be obtained by the conventional microscope but also the third-dimensional information in the optical axis direction are obtained. It is possible to observe the specimen three-dimensionally. For these reasons, confocal microscopes have been rapidly used in recent years, where there is a demand for observing the three-dimensional structure of a specimen.

However, in principle, the confocal microscope irradiates the surface of the sample with spot-like illumination light (light spot), and two-dimensionally scans the surface of the sample while measuring the light intensity in synchronization therewith. There is a need to. For this reason, a laser, a relay lens, a deflection optical element, a Nipkow disk, or a disk rotating mechanism is required as an optical system for irradiating a sample with a light spot. As a result, there is a problem that the entire microscope system tends to be large and the manufacturing cost is high.

On the other hand, as a small-sized microscope capable of inexpensively obtaining a three-dimensional image of a sample, there is a microscope that illuminates the sample in a lattice shape (hereinafter referred to as a lattice illumination microscope). In this grating illumination microscope, the signal intensity of the 0th-order light hardly changes with the defocus in the illumination light that is illuminated on the sample and scattered here, but the light other than the 0th-order light is rapidly defocused. It is made with a focus on losing signal strength. That is, in the lattice illumination microscope, the specimen is illuminated in a lattice pattern and the main component of scattered light is intentionally set to 0.
The microscope is sensitive to defocusing by removing the remaining zero-order light by performing an inter-image calculation by setting the light to be other than the next light. Therefore, a three-dimensional image of the sample can be obtained by moving the sample in the optical axis direction to obtain a signal from only the in-focus surface of the sample using the grating illumination microscope. .

The principle of this grating illumination microscope will be briefly described. First, assuming a one-dimensional sine wave of the pitch Λ as the illumination pattern of the sample, the relationship of Expression (1) is established, and therefore the light intensity I (t, ω) at each point on the image is calculated by Expression (2). become that way.

[0007]

[Equation 1] S (t, ω) = 1 + m ・ cos (ν ・ t + φ) (1) However, ν = λ / (Λ · n · NA)

[0008]

## EQU2 ## I (t, ω) = I ₀ + I _c cosφ ₀ + I _s sinφ ₀ (2)

In the equation (1), S (t, ω) is the standard
It is a light intensity distribution on a book. (t, ω) is the actual coordinate on the sample
Normalize (x, y) with wavelength λ, refractive index n, and numerical aperture NA
Coordinates (t, ω) = (2πnNA / λ) · (x, y)
The relationship is established. The symbol m indicates the amplitude of the sine wave, and m is
The larger the sine-wave illumination, the higher the contrast.
Means ν is the illumination pattern projected on the sample
Of the pitch Λ and the refractive index n between the objective lens and the sample
Spatial frequency. NA is the numerical aperture, NA = sin α
Where α is the chief ray and the light that passes through the outermost side of the objective lens.
Is the angle formed by. φ is the origin of sinusoidal illumination
This is a phase, and this microscope captures three images in three phases.
Get, but φ₀Is the first phase. Therefore,
Φ shown below ₁Is the second phase, φ_TwoIs the third phase
It

Here, φ ₀ = 0, φ ₁ = 2π / 3, φ ₂
= 4π / 3, Square-Law in communication engineering
Similarly to, the 0th-order light can be removed by the expression (3). As a result, the signal response to the defocus at this time is expressed by Expression (4). Therefore, the resolution of the optical system in the optical axis direction is approximately as shown in equation (5). In these equations, J1 is a first-order Bessel function, λ is a light wavelength, and n is a refractive index between the objective lens and the sample.

[0011]

[Equation 3]

[0012]

[Equation 4]

[0013]

[Equation 5]

Thus, according to the equation (5), it is possible to make the resolution Δz in the optical axis direction a very small value by selecting an appropriate ν, and in that case, it becomes sensitive to defocus. I understand. Therefore, in the observation with this microscope, the focus is achieved only in the region of the extremely small width in the optical axis direction, and the observation sample is obtained by moving the observation sample in the optical axis direction in such a focused state. A three-dimensional image of the observation sample can be obtained by processing the observation image according to the amount of movement in the optical axis direction.

An example of a grating illumination microscope constructed according to the above principle is shown in FIG. This microscope has a one-dimensional grating 53 having a sinusoidal transmittance distribution that changes in the direction of arrow A. The one-dimensional grating 53 makes light emitted from a white light source 51 into a substantially parallel light flux by a condenser lens 52. It is illuminated by the light. As a result, light having a one-dimensional lattice pattern (striped pattern) whose light intensity changes sinusoidally in the one-dimensional direction is emitted from the one-dimensional lattice 53, passes through the illumination lens 56, and is observed by the half mirror 57 under a microscope. It is incident on the optical path.

In this optical path, an objective lens 58 located below the half mirror 57, a sample table 60 disposed below the objective lens 58, and a relay lens located above the half mirror 57. 61 and a CCD camera (imaging element) 62 located above the relay lens 61 are arranged side by side on the same optical axis extending vertically. The light of the one-dimensional lattice pattern emitted from the one-dimensional lattice 53 and transmitted through the illumination lens 56 as described above is reflected by the half mirror 57, transmitted through the objective lens 58 that shares the pupil position with the illumination lens 56, and the sample. An image is formed on the sample mounting surface 60a of the table 60. That is, the position of the one-dimensional grating 53 with respect to the illumination lens 56 and the sample mounting surface 60 with respect to the objective lens 58.
The position of a is set in a conjugate relationship, and the sample table 60
An image of the one-dimensional lattice 53 is formed on the sample mounting surface 60a. As a result, the sample mounting surface 60 of the sample table 60
The sample placed on a is illuminated by light of a one-dimensional lattice pattern having an intensity distribution that is made by transmitting through the one-dimensional lattice 53 and changes in a one-dimensional sinusoidal shape.

The image of the sample illuminated by the one-dimensional lattice pattern illumination in this manner is used for the objective lens 58 and the relay lens 61.
An image is formed on the CCD camera 62 through the image forming optical system, and the signal is taken into the control computer 63 to obtain a lattice sample image.

Next, the control computer 63 outputs a control signal to the piezoelectric element driver 55, the piezoelectric element driver 55 slightly expands and contracts the piezoelectric element 54, and the one-dimensional lattice 53 connected to this piezoelectric element 54 is formed into a lattice. It is moved in the direction of arrangement (the direction of arrow A). At this time, the piezoelectric element 54
The expansion / contraction length of 1 / the grid pitch of the one-dimensional grid 53
Set it to 3. Then, the lattice pattern (stripes) illuminated on the sample on the sample mounting surface 60a has a phase of 1/3.
The pattern will be shifted. Then, the sample image at this time is picked up by the CCD camera 62 as in the above.

Similarly, the piezoelectric element 54 moves the one-dimensional grating 53 by 2/3 of the grating pitch to illuminate the sample, and the sample image at this time is also taken by the CCD camera 62.

When the light intensities I _p are calculated by the equation (3) using I ₁ , I ₂ , and I ₃ in the above equation (3) as the images picked up as described above, the light intensity I _p on the sample is calculated. Only the image at the in-focus height can be obtained.
A three-dimensional image of the sample can be obtained by moving the sample table 60 in the optical axis direction and capturing an image at a focused height in the same manner as above.

By the way, in the grating illumination microscope shown in FIG. 4, the vertical resolution is ν as shown in equation (5).
= 1, that is, the highest when Λ = λ / nNA. Since the resolution limit in a microscope is theoretically λ / 2nNA, the above illumination pitch Λ is only twice the lower limit that can be resolved by a microscope, and is quite fine. Since the CCD camera 62 captures an image of the sample that has been subjected to such fine grid-like illumination, the pitch of the grid-like illumination captured by the CCD camera 62 is also quite small. For example, the wavelength λ
= 546 nm illumination light and NA = 0.7 objective lens are combined, the grating illumination pitch that gives the highest vertical resolution is Λ = 0.546 / 0.72 on the sample.
= 0.755 μm. When the CCD camera 62 takes an image of this lattice illumination, the objective lens 58 and the relay lens 6
If the total magnification of 1 is 40 times, a grid pattern with a pitch of about 30 μm is formed on the CCD camera 62.

[0022]

By the way, general C
Since the pixel size of the CD camera 62 is about 10 μm, one cycle of the above grid-like illumination is the CCD camera 62.
It is only 3 pixels. In such a situation, when a CCD camera captures an image of a sample illuminated by a lattice pattern, the lattice pattern imaged by the CCD camera 62 can be obtained by only slightly shifting the lattice pattern pitch from an integer multiple of the pixel pitch. There is a problem that imbalance occurs in the contrast of the pattern. This will be described below.

In the above-mentioned grating illumination microscope, the signal intensity of each CCD pixel (pixel) when the sinusoidal grating illumination is imaged by the CCD camera 62 is shown in FIGS. (Note that Fig. 5
(B) is an enlarged view of a part of FIG. 5 (a). However, at this time, when the CCD camera takes an image, the period of the grating illumination on the CCD image pickup device is about 3.02.
The case where it becomes a pixel is shown. As can be seen from these figures, even if the sample is given grid-shaped illumination with a sinusoidal intensity distribution, the period of the sinusoidal illumination (grid pitch)
Is not sufficiently large with respect to the pixel size of the image sensor (CCD camera), it is difficult to accurately capture and reproduce the intensity distribution of the sinusoidal illumination.

In particular, when the pitch of the grid-like illumination is slightly deviated from an integer multiple of the pixel arrangement pitch, it is difficult to accurately image the intensity distribution of the sinusoidal illumination, and the pitch A phenomenon in which the intensity distribution fluctuates at a large pitch according to the shift (a phenomenon similar to the so-called moire phenomenon, and as shown in FIG. 5, when the intensity changes in a cycle of approximately three pixels (pixels). There is a problem in that the intensity of each three-pixel interval changes into a large waviness) and the contrast becomes non-uniform as a whole. As a result, there is a problem in that not only the S / N of the image captured by the CCD camera is not uniform, but also the image after the image processing by the above-mentioned formula (3) has uneven density. Such a problem reduces the evaluation accuracy of the sample.

As can be seen from this, when the pitch of the grid pattern for illuminating the sample is reduced in order to obtain high resolution in the grid illumination microscope, the pitch of the grid pattern becomes too small with respect to the pixel pitch of the CCD camera, and It becomes impossible to use a sufficient number of pixels for the checkerboard pattern,
On the contrary, the vertical resolution will be lowered. To solve this problem, by increasing the focal length of the relay lens, C
It is considered that the pitch of the lattice pattern on the CD camera should be widened so that a sufficient number of pixels can be used for each lattice pattern. However, in this case, there is a problem that a sufficient observation field of view cannot be obtained unless the image pickup surface of the image sensor is increased, and it is costly to make a large image sensor capable of obtaining a sufficient observation field of view. There is a problem that it is not realistic from the point of view.

The present invention has been made in view of these problems.
Provided is a grating illumination microscope having a configuration capable of picking up a good image without enlarging the image pickup surface of an image pickup element even when the pitch of a lattice pattern for illuminating a sample is small, and an observation method using the same. The purpose is to

[0027]

In order to achieve such an object, a grating illumination microscope according to the present invention provides an illumination light source for emitting illumination light, a sample stand for mounting a sample, and an illumination light source for emitting light. The illumination optical system that illuminates the sample on the sample table with the illuminated illumination light and the observation light emitted from the sample on the sample table by the illumination of the lattice pattern to form the image of the sample And an image pickup device for picking up an image of the sample formed by the image pickup optical system,
It has a direction adjusting means for adjusting the positions of the direction of the lattice pattern in the image of the sample taken by the image pickup device and the arrangement direction of the pixels in the image pickup device.

According to the grating illumination microscope of the present invention having such a configuration, the direction adjusting means adjusts the positions of the direction of the lattice pattern in the image of the sample taken by the image sensor and the arrangement direction of the pixels in the image sensor. By this adjustment, it is possible to adjust the contrast by changing the relationship between the pitch of the lattice pattern on the image pickup surface of the image pickup device and the pixel pitch forming the image pickup device. This allows
As described above, when an image signal captured by the image sensor is processed to obtain an in-focus image in a region having a very small width in the optical axis direction, uneven density of this image is suppressed and the image quality is reduced. Can be improved.

The direction adjusting means is preferably constructed so as to rotate the image pickup device in a plane perpendicular to the optical axis.

The lattice illumination microscope according to the second aspect of the present invention includes an illumination light source that emits illumination light, a sample table on which a sample is placed, and an illumination light beam emitted from the illumination light source to a sample on the sample table. An illumination optical system for illuminating a lattice pattern, an imaging optical system for receiving an observation light emitted from a sample on a sample table upon receiving the illumination of the lattice pattern, and an image of the sample, and an imaging optical system And an image pickup device for picking up an image of the sample formed by the image pickup device, and the direction of the lattice pattern in the image of the sample taken by the image pickup device and the arrangement direction of pixels in the image pickup device are set so as not to overlap with each other. ing.

By setting such that the direction of the lattice pattern in the image of the sample taken by the image pickup device and the arrangement direction of the pixels in the image pickup device do not overlap with each other in this way, the imbalance of the contrast in the image can be suppressed, and the image pickup When an image signal picked up by an element is processed to obtain an image in focus in a region with a very small width in the optical axis direction, it is possible to suppress the occurrence of uneven density in the image and improve the image quality. it can.

The angle formed by the direction of the lattice pattern in the image of the sample taken by the image pickup device and the direction in which the pixels are arranged in the image pickup device is set to 20 ° to 70 °, which causes unevenness in image density. It is desirable for suppressing the occurrence and improving the image quality.

In the observation method using the grating illumination microscope according to the present invention, the sample to be observed is placed on the sample table, the illumination light having a lattice pattern is emitted from the illumination light source, and the illumination emitted in this manner is used. The illumination optical system guides the light onto the sample table to illuminate the sample in a grid pattern, and the observation light emitted from the sample upon receiving the grid pattern illumination is guided to the image sensor by the imaging optical system and It is configured to take an image of the specimen. When the image of the sample is captured by the image sensor in this way, the position of the lattice pattern in the image of the sample captured by the image sensor does not overlap the direction in which the pixels of the image sensor are arranged. Is adjusted to shoot.

[0034]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below with reference to the drawings. The configuration of the grating illumination microscope according to the embodiment of the present invention is shown in FIG. This configuration is similar to the configuration of the conventional reflection type grating illumination microscope shown in FIG. 4, and has a one-dimensional grating 3 having a sinusoidal transmittance distribution that changes in the direction of arrow A. Lattice 3
Is illuminated by the light emitted from the white light source 1 by the condenser lens 2 into a substantially parallel light flux. As a result, illumination light having a one-dimensional lattice pattern (striped pattern) whose light intensity changes sinusoidally in the one-dimensional direction is emitted from the one-dimensional lattice 3. Then, the illumination optical system is configured so that the illumination light passes through the illumination lens 4 and is incident on the microscope observation optical path (including the imaging optical system) by the half mirror 5. By using the one-dimensional grating, the structure of the grating illumination microscope is the simplest and the three-dimensional observation is the easiest.

The one-dimensional lattice 3 is supported by the piezoelectric element 11. The piezoelectric element 11 is actuated by the piezoelectric element driver 10 which receives a control signal from the control computer 9 to slightly expand and contract, so that the one-dimensional lattice 3 can be moved in the arrow A direction.

In the microscope observation optical path, the objective lens 6 located below the half mirror 5 and the objective lens 6
On the same optical axis in which a sample table 15 arranged below the relay mirror 7, a relay lens 7 located above the half mirror 5, and a CCD camera (imaging device) 8 located above the relay lens 7 extend vertically. Are arranged side by side. The illumination light having the one-dimensional lattice pattern emitted from the one-dimensional lattice 3 and transmitted through the illumination lens 4 as described above is reflected by the half mirror 5, passes through the objective lens 6, and is irradiated toward the sample table 15. .

As described above, the illumination light transmitted through the objective lens 6 and applied to the sample table 15 forms a lattice image of the one-dimensional grating 3 on the upper surface of the sample table 15. That is, the position of the one-dimensional grating 3 with respect to the illumination lens 4 and the position of the sample mounting surface 15a with respect to the objective lens 6 are set in a conjugate relationship, and the one-dimensional grating 3 on the sample mounting surface 15a of the sample table 15.
You can make a statue of. As a result, the sample 16 mounted on the sample mounting surface 15a is illuminated by a one-dimensional lattice pattern having an intensity distribution that is formed by transmitting through the one-dimensional lattice 3 and changes in a one-dimensional sinusoidal shape. . Accordingly, the image of the lattice member can be formed on the sample mounting surface 15a of the sample table 15, and the sample 16 placed on the sample mounting surface 15a.
Can be illuminated in a checkerboard pattern.

The reflected light from the sample thus illuminated by the lattice pattern is condensed through the image forming optical system consisting of the objective lens 6 and the relay lens 7, is incident on the CCD camera 8, and is imaged by the CCD camera 8. , The signal is taken into the control computer 9 and stored.

Next, the control computer 9 outputs a control signal to the piezoelectric element driver 10, and the piezoelectric element driver 1
Control to slightly expand and contract the piezoelectric element 11 from 0,
The one-dimensional lattice 3 connected to the piezoelectric element 11 is moved in the direction in which the lattices are arranged (the direction of arrow A). At this time, the expansion / contraction length of the piezoelectric element 11 is set to be 1/3 of the lattice pitch of the one-dimensional lattice 3. Then, the sample mounting surface 15
The phase of the lattice pattern (stripe) illuminated on the sample on a is 1
The pattern is shifted by / 3. Then, the sample image at this time is picked up by the CCD camera 8 similarly to the above, and the signal thereof is taken in the control computer 9 and stored therein.

Similarly, the piezoelectric element 11 moves the one-dimensional grating 3 by 2/3 of the grating pitch to illuminate the sample, and the sample image at this time is also captured by the CCD camera 8 to control the signal. It is taken into the computer 9 and stored.

If the light intensities I _p are calculated according to the equation (3) by using I ₁ , I ₂ , and I ₃ in the above equation (3) as the images picked up as described above, then the light intensity I _p on the sample is calculated. Only the image at the in-focus height can be obtained.
Then, the sample table 15 is moved in the optical axis direction by the sample table moving mechanism 17 to capture an image at the focused height in the same manner as above. Sample stage moving mechanism 1 at this time
The movement information by 7 is input to the control computer 9,
By performing image processing by combining the movement information and the image obtained as described above, it is possible to obtain a three-dimensional image of the sample mounted on the sample mounting surface 15a.

As described above, when the image of the sample is obtained by the grating illumination microscope having the structure shown in FIG. 1, the one-dimensional lattice 3 has a small lattice pitch and the sample 16 illuminated by the lattice pattern is CCD camera. If the pixel pitch is not very small with respect to the pitch of the lattice pattern on the imaging surface when the image is picked up by No. 8 (for example, the pitch of the lattice pattern is about several times the pixel pitch), as shown in FIG. 5 described above. There is a problem that a large undulation-like change in signal intensity occurs, an imbalance occurs in the contrast of the image, and unevenness in the image density occurs. When the contrast in the image is calculated based on the calculation similar to that of FIG. 5 shown in the prior art, the good contrast part is 1.16, while the poor contrast part is 1.00, and the contrast as a whole is high. Was uneven.

In order to correct such a contrast imbalance, in the grating illumination microscope of FIG. 1, C is used.
A direction adjusting device 12 for rotating the CD camera 8 about the light receiving optical axis as shown by an arrow B is provided. That is, the direction adjusting device 12 can rotate the image pickup surface of the CCD camera 8 about the optical axis without changing the image pickup surface in the optical axis direction. As a result, the contrast could be made uniform to 1.10 as a whole.

The maximum contrast is obtained when the pitch of the lattice pattern is an integral multiple of the pixel pitch. These contrasts have three phases (φ ₀ = 0, φ ₁ = 2π / 3, φ ₂ = 4π / 3) for each pixel.
The light intensity (I ₀ , I ₁ , I ₂ ) at is calculated by the equation (6). This is the value of (I _i −I _j ) calculated by the above equation (3) with respect to the average light intensity for all three combinations,
It is an average.

[0045]

[Equation 6]

FIG. 2A shows an image obtained by the CCD camera 8 of the sample 16 which is illuminated by the lattice when the direction of the lattice pattern and the direction of the pixel are adjusted by the direction adjusting device 12. Shows. In this case, the grid illumination extends in the vertical direction and the brightness in the horizontal direction periodically changes, but the overall brightness is bright on the left side and dark on the right side. this is,
As shown in FIG. 5, a phenomenon occurs in which the signal intensity of a pattern that periodically changes corresponding to the pitch of the grid illumination changes into a large undulation, and such a brightness (contrast) is generated.
It is thought that there is an imbalance.

Then, the CCD camera 8 is adjusted to rotate by the direction adjusting device 12, and the image taken by tilting the lattice pattern direction by 30 degrees with respect to the pixel direction is shown in FIG. 2B.
2 (c) shows an image captured with a tilt of 45 degrees, and FIG. 2 (d) shows an image captured with a tilt of 60 degrees. As can be seen from these figures, the contrast of the image is highest in FIG. 2B, and deteriorates in the order of FIG. 2D and FIG. 2C. In this way, it can be seen that when the direction of the grid pattern is tilted with respect to the direction of the pixels, the unbalance of the brightness as a whole becomes small. Although 30 ° and 60 ° have a symmetrical positional relationship with respect to each other with respect to 45 °, the contrast states of FIG. 2 (b) and FIG. 2 (d) are different from those of FIG. 2 (c). Although not symmetrical, this is because the CCD camera 8 has a rectangular shape in which the vertical and horizontal sizes of each pixel are different, and the window size of each pixel is different vertically and horizontally. If the cell shape and the window shape of the CCD camera 8 are square, then 4
5 ° is the best contrast.

As a result of various experiments examining the relationship between the inclination and the imbalance of the brightness of the image, the brightness unbalance is caused when the direction of the lattice pattern is inclined by 20 to 70 degrees with respect to the arrangement direction of the pixels. It turns out that the effect of reducing the balance is great. Therefore, in the device of FIG.
Although the CCD camera 8 is rotated by the above, the direction adjusting device 12 is not used, and the one-dimensional lattice 3 is arranged so that the direction of the lattice pattern is 20 to 70 degrees with respect to the pixel arrangement direction. The installation position may be set. In that case, a direction adjusting device is provided on the one-dimensional lattice 3 and the one-dimensional lattice 3 is rotated.

[0049]

EXAMPLES Specific observations were made using the grating illumination microscope having the configuration shown in FIG. At this time, the pixel size of the CCD camera 8 is 9.8 μm in the vertical direction and 8.4 μm in the horizontal direction.
As the surface of the IC chip, an aluminum wiring layer was used. Then, the result when the image of the surface of the IC chip is obtained by matching the direction of the grid illumination and the pixel array direction is shown in FIG.
FIG. 3B shows the result when the image on the surface of the IC chip is obtained by inclining the pixel arrangement direction by 60 degrees with respect to the direction of the grid illumination, as shown in FIG. As can be seen by comparing the two figures, there is an imbalance in contrast in the image when the grid illumination direction and the pixel array direction match, but the pixel array direction is different from the grid illumination direction. This imbalance is greatly improved in the image tilted by 60 degrees. Due to the relationship between the accuracy of phase shift for one stripe and the uneven intensity distribution (contrast unevenness) on the CCD,
Uneven intensity on the image (black and whitish areas)
Are appearing alternately.

[0050]

As described above, according to the present invention,
The rotation position between the direction of the lattice pattern in the image of the sample taken by the image pickup device and the arrangement direction of the pixels in the image pickup device can be adjusted by the direction adjusting means, and by this adjustment, the pitch of the lattice pattern on the image pickup surface of the image pickup device. The contrast can be adjusted by changing the relationship between the pixel pitch of the image sensor and the pixel pitch of the image sensor. With this, when processing the image signal picked up by the image pickup device to obtain a three-dimensional image or the like, it is possible to suppress the occurrence of uneven density in the image and improve the image quality.

According to the second aspect of the present invention, the direction of the lattice pattern in the image of the sample photographed by the image pickup device and the arrangement direction of the pixels in the image pickup device do not overlap, more specifically, the image pickup by the image pickup device is performed. Since the angle between the direction of the lattice pattern in the sampled image and the direction in which the pixels are arranged in the image sensor is set to 20 degrees to 70 degrees, the image signal captured by the image sensor is processed. It is possible to improve the image quality by suppressing the occurrence of unevenness in the density of the obtained image.

In the observation method using the grating illumination microscope according to the present invention, the sample to be observed is placed on the sample table, the illumination light having a lattice pattern is emitted from the illumination light source, and the illumination emitted in this manner is used. The illumination optical system guides the light onto the sample table to illuminate the sample in a grid pattern, and the observation light emitted from the sample upon receiving the grid pattern illumination is guided to the image sensor by the imaging optical system and It is configured to capture an image of the specimen, and when the image of the specimen is captured by the image sensor in this manner, the direction of the lattice pattern in the image of the specimen captured by the image sensor and the arrangement of pixels in the image sensor. The photographing is performed by adjusting the rotation position with respect to the vertical direction, and in this case also, similarly to the above, it is possible to suppress the occurrence of uneven density in the image and improve the image quality.

[Brief description of drawings]

FIG. 1 is a schematic diagram illustrating a configuration of a grating illumination microscope according to the present invention.

FIG. 2 is a diagram showing images captured by the lattice illumination microscope when a pixel direction and a lattice pattern direction are aligned with each other, when tilted by 30 degrees, when tilted by 45 degrees, and when tilted by 60 degrees. Is.

FIG. 3 is a diagram showing an image of a sample obtained by performing image processing in a case where the direction of a pixel and a direction of a grid pattern are matched and when the direction is tilted by 60 degrees in the grid illumination microscope. .

FIG. 4 is a schematic diagram illustrating a configuration of a conventional grating illumination microscope.

FIG. 5 is a graph showing a change in signal intensity for each pixel when an image of a sample lattice-illuminated by a lattice illumination microscope is captured by a CCD camera.

[Explanation of symbols]

3 one-dimensional lattice 4 Lighting lens 5 half mirror 6 Objective lens 7 relay lens 8 CCD camera 9 Control computer 11 Piezoelectric element 12 Rotation adjustment device 15 specimen table

Claims (5)

[Claims] 1. An illumination light source that emits illumination light, a sample table on which a sample is placed, and an illumination that causes the sample on the sample table to be illuminated with a lattice pattern by the illumination light emitted from the illumination light source. An optical system; an image-forming optical system that receives the observation light emitted from the sample on the sample table upon receiving the illumination of the lattice pattern to form an image of the sample; and an image formed by the image-forming optical system. And an image pickup device for picking up an image of the sample, and adjusting the position of the lattice pattern direction in the image of the sample picked up by the image pickup device and the arrangement direction of pixels in the image pickup device. A grating illumination microscope having a direction adjusting means. 2. The grating illumination microscope according to claim 1, wherein the direction adjusting unit is configured to rotate the image pickup device in a plane perpendicular to the optical axis. 3. An illumination light source that emits illumination light, a sample table on which a sample is placed, and an illumination that causes the sample on the sample table to perform a grid pattern illumination by the illumination light emitted from the illumination light source. An optical system; an image-forming optical system that receives the observation light emitted from the sample on the sample table upon receiving the illumination of the lattice pattern to form an image of the sample; and an image formed by the image-forming optical system. And an image pickup device for picking up an image of the sample, and the setting is made so that the direction of the lattice pattern in the image of the sample picked up by the image pickup device and the arrangement direction of pixels in the image pickup device do not overlap. Lattice illumination microscope characterized in that 4. The angle formed by the direction of the lattice pattern in the image of the sample taken by the imaging device and the direction in which the pixels of the imaging device are arranged is 20 degrees to 70 degrees. The grating illumination microscope according to claim 3. 5. A sample to be observed is placed on a sample table, illumination light having a lattice pattern is emitted from an illumination light source, and the illumination light thus emitted is guided onto the sample table by an illumination optical system. The sample is illuminated with the lattice pattern, and the observation light emitted from the sample upon receipt of the lattice pattern illumination is guided to an image sensor by an imaging optical system, and an image of the sample is captured by the image sensor. When the image of the sample is captured by the image sensor in this way, the direction of the lattice pattern in the image of the sample captured by the image sensor and the pixels in the image sensor. An observation method using a grating illumination microscope, characterized in that the position is adjusted so as not to overlap the direction in which the images are arranged and the image is taken.