JP2003015048A - Lattice illumination microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To use a microscope which illuminates a specimen in a lattice shape also as a fluorescence microscope. SOLUTION: The lattice illumination microscope is provided with an illumination optical system to emit excitation illumination light with one dimensional lattice pattern from one dimensional lattice 3, a specimen stand 15 having a specimen placing surface 15a for placing the specimen 16 which emits fluorescence in response to the excitation illumination light, an objective lens 8 arranged above the specimen stand 15, an imaging optical system which forms the image of the specimen in a CCD camera 8 in response to the fluorescence emitted from the specimen 16 placed on the specimen placing surface 15a through the objective lens 6, and a position adjuster 20 to position the one dimensional lattice 3. The position adjuster 20 is provided with a micrometer caliper mechanism 22 to move the one dimensional lattice 3 in an optical axis direction. The micrometer caliper mechanism 22 moves the one dimensional lattice 3 in the optical axis direction and corrects focus displacement caused by a wavelength difference between the exciting light and the fluorescence.

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 that illuminates a specimen in a lattice pattern.

[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.

[0021]

By the way, the inventor of the present invention considered using the grating illumination microscope having the structure shown in FIG. 3 as a fluorescence microscope. In this case, the white light source 5
Instead of 1, an excitation light source is used, and the excitation light emitted from this excitation light source is applied to the one-dimensional lattice 53 through the condenser lens 52 to produce one-dimensional lattice pattern of excitation light.
Then, the excitation light of the one-dimensional pattern is made incident from the illumination lens 56 through the half mirror 57 into the optical path of the microscope observation, and the sample 60 on the sample table 60 is passed through the objective lens 58.
Irradiate a. This sample 60a emits fluorescence upon receiving the irradiation of the excitation light, and this fluorescence is received by the CCD camera 62 through the objective lens 58 and the relay lens 61, and the sample 60a
A fluorescent image of a is obtained. By processing this in the same manner as above, a three-dimensional image based on the fluorescence of the specimen can be obtained.

When the grating illumination microscope of FIG. 3 is used as a fluorescence microscope in this way, the half mirror 57 is used.
Is composed of a dichroic mirror having a high reflectance for the wavelength of the excitation light and a high transmittance for the wavelength of the fluorescence. As a result, the sample 60a is illuminated with excitation light in a one-dimensional lattice pattern, and the CCD camera 62 can capture an image of the sample viewed at the fluorescence wavelength emitted by receiving the excitation light illumination. A three-dimensional image of the sample is obtained by performing arithmetic processing by.

By the way, when the grating illumination microscope is used as a fluorescence microscope as described above, the objective lens 58 is constructed so that excitation light and fluorescence pass therethrough, and the axial chromatic aberration of the objective lens 58 for these lights poses a problem. Become. This will be specifically described. In a normal grating illumination microscope, the reflected light of the illumination light applied to the sample is received by the CCD camera 62, and the illumination light passing through the objective lens 58 and the reflected light have the same wavelength. However, in the case of a fluorescence microscope, since light of different wavelengths such as excitation light and fluorescence pass through, axial chromatic aberration of the objective lens 58 with respect to these lights occurs. Therefore, when the one-dimensional lattice image by the excitation light is accurately formed on the sample, there is a problem that the image based on the fluorescence from this sample is not accurately formed on the CCD camera 62.

This occurs because the focal position is displaced due to the axial chromatic aberration of the objective lens, and the focal position of the illumination optical system is adjusted so that a one-dimensional lattice image by the excitation light is accurately formed on the sample. At the same time, it is considered that the problem can be solved by adjusting the focus position of the imaging optical system so that the sample image by fluorescence is accurately formed on the CCD camera. However, in order to use a normal grating illumination microscope as a fluorescence microscope, such adjustment is necessary each time, and further,
There is a problem that such adjustment is required every time the combination of excitation light and fluorescence used in the fluorescence microscope is different, and it is difficult to use the grating illumination microscope having the above-mentioned configuration as the fluorescence microscope.

The present invention has been made in view of these problems.
It is an object of the present invention to allow a grating illumination microscope to be easily used as a fluorescence microscope.

[0026]

In order to achieve such an object, in the present invention, an illumination light source for emitting illumination light,
A sample table on which the sample is placed, an objective lens arranged above the sample table, and illumination light emitted from an illumination light source through the objective lens to illuminate the sample on the sample table in a lattice pattern. A grating illumination microscope is configured with an optical system and an imaging optical system that forms an image of a sample by receiving observation light emitted from a sample on a sample table through an objective lens by receiving illumination in a lattice pattern. , The wavelengths of the illumination light and the observation light are different, and the focus of the illumination optical system at the wavelength of the illumination light matches and the focus of the imaging optical system at the wavelength of the observation light matches. It has a focus correction means for adjusting the focus of at least one of the image optical systems.

In the grating illumination microscope according to the present invention,
When using a general grating illumination microscope that forms and observes reflected light from a sample using an ordinary white light source as the illumination light as it is as a fluorescence microscope, the wavelength of the excitation light used as the illumination light by the focus correction means and The focus position may be adjusted according to the difference with the wavelength of the fluorescence emitted from the sample.

The grating illumination microscope having the above-mentioned configuration can be constructed by providing an optical path changing optical element that causes the illumination light transmitted through the illumination lens to enter the optical path of the imaging optical system and irradiate it toward the objective lens.

The focus correction means is configured to adjust the focus position of the illumination optical system so as to focus the illumination of the lattice pattern on the sample on the sample table by the illumination optical system. You may.

In this case, the illumination optical system is combined with an objective lens and a grating member that has a transmittance distribution corresponding to the lattice pattern and emits transmitted light as illumination light of the lattice pattern, and receives the light of the lattice pattern. And an illumination lens for projecting a lattice pattern image on the sample placed on the sample table,
The focus correction means may be composed of a grating position adjusting means for adjusting the position of the grating member in the optical axis direction.

The grid position adjusting means comprises a grid moving mechanism for moving the grid member in the axial direction and a grid operating member for externally operating the grid moving mechanism, and the grid operating member is illuminated with illumination light. It is preferable to provide a mark indicating the operation position corresponding to the difference in wavelength between the observation light and the observation light.

Further, the focus correction means may be composed of a correction optical element for an illumination optical system which is arranged in the optical path of the illumination optical system and adjusts the focus position of the illumination optical system. In this case, the correction optical element for the illumination optical system can be removed and replaced,
It is preferable to exchange the illumination light and the observation light according to the type and the type of the objective lens.

The focus correction means adjusts the focus position of the imaging optical system so as to focus the image of the sample upon receiving the observation light emitted from the sample on the sample table by the imaging optical system. Can be configured to do so.

In this case, the focus correction means may be composed of a correction optical element for the image forming optical system which is arranged in the optical path of the image forming optical system and adjusts the focus position of the image forming optical system. In this configuration, it is preferable that the correction optical element for the imaging optical system is detachable and replaceable, and that the correction optical element is replaced according to the types of illumination light and observation light and the type of objective lens.

Further, an image pickup device for picking up an image of a sample formed by the image forming optical system is provided, and the focus correction means is constituted by an image pickup element position adjusting means for adjusting the position of the image pickup element in the optical axis direction. good.

In this case, the image pickup device position adjusting means comprises an image pickup device moving mechanism for moving the image pickup device in the axial direction and an image pickup device operating member for externally operating the image pickup device moving mechanism. It is preferable that the operation member is provided with a mark indicating an operation position corresponding to a difference in wavelength between the illumination light and the observation light.

[0037]

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 first embodiment of the present invention is shown in FIG.
This structure is similar to the structure of the conventional reflection type grating illumination microscope shown in FIG. 3, and has a one-dimensional grating 3 having a sinusoidal transmittance distribution that changes in the direction of arrow A. The grating 3 illuminates the excitation light emitted from the excitation light source 1 with the light made into a substantially parallel light flux by the condenser lens 2. As a result, the excitation 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 such that the excitation 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 further, the three-dimensional observation is the easiest.

The one-dimensional lattice 3 is supported by the position adjusting device 20. The position adjusting device 20 is connected to the one-dimensional grating 3 and moves the one-dimensional grating 3 in the transmittance distribution change direction of the grating (direction of arrow A),
The piezoelectric element 21 is moved in the optical axis direction (arrow B direction) to move the one-dimensional grating 3 in the optical axis direction (arrow B direction). Piezoelectric element 2
1 is operated by a piezoelectric element driver 10 which receives a control signal from a control computer 9 to slightly expand and contract to move the one-dimensional lattice 3 in the arrow A direction.
Further, the micrometer mechanism 22 rotates the adjustment knob 22a to move the piezoelectric element 21 in the optical axis direction (arrow B).
Direction) to move the one-dimensional grating 3 in the optical axis direction (arrow B direction).

In the microscope observation optical path, the objective lens 6 located below the half mirror 5 and the objective lens 6 are provided.
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 excitation illumination light having a 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, transmitted through the objective lens 6, and irradiated toward the sample table 15. It As you can see from this, the half mirror 5
Is composed of a dichroic mirror having a high reflectance characteristic with respect to the excitation light wavelength.

As described above, the excitation illumination light that has passed through the objective lens 6 and irradiated on 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 objective lens 6
The position of the sample mounting surface 15a with respect to is set in a conjugate relationship, and an image of the one-dimensional lattice 3 can be formed on the sample mounting surface 15a of the sample table 15. As a result, the sample placed on the sample placing surface 15a is illuminated by the excitation light of the one-dimensional lattice pattern having the intensity distribution which is created by transmitting through the one-dimensional lattice 3 and changes in a one-dimensional sinusoidal shape. Receive. As a result, the image of the lattice member is displayed on the sample mounting surface 15a of the sample table 15.
The sample placed on the sample mounting surface 15a can be illuminated in a lattice pattern.

The sample has a fluorescent substance, and the excitation light source 1
When the excitation light from the sample is received, fluorescence is emitted, and thus the fluorescence emitted from the sample is condensed through an image forming optical system including the objective lens 6 and the relay lens 7 to form an image of the sample on the CCD camera 8. The signal is taken into the control computer 9 to obtain an image of interference fringes. Therefore, the half mirror 5 is composed of a dichroic mirror having a high transmittance characteristic with respect to the fluorescence wavelength. That is, the half mirror 5 is composed of a dichroic mirror having a characteristic of reflecting excitation light and transmitting fluorescence.

Here, since the excitation light and the fluorescence have different wavelengths, the axial chromatic aberration in the objective lens 6 through which these lights pass is different. For this reason, the image of the sample taken by the CCD camera 8 is displayed on the monitor 11 connected to the control computer 9, and the focus adjustment of the imaging optical system (the focus adjustment normally performed in the microscope, for example,
The sample mounting surface 15 is adjusted by moving the sample table up and down.
When the fluorescent image of the sample placed on a is made to be clearly visible, the lattice pattern irradiated on the sample is often out of focus and in a blurred state.

From this state, if the adjustment knob 22a of the position adjusting device 20 is operated to move the one-dimensional grating 3 in the direction of the optical axis (direction of arrow B), the fluorescence image of the specimen changes sharply. Without this, there is a lattice pattern focus, and the lattice image is superimposed on the sample image. This is because the shift of the focal position due to the axial chromatic aberration is corrected by the adjustment by the position adjusting device 20, and the lattice image on the sample by the excitation light is exactly the same as the surface on the sample observed by the CCD camera 8. This is because it becomes a face. Therefore, the position of the one-dimensional lattice 3 is fixed at this position. Therefore, it is preferable to provide the micrometer mechanism 22 with means for fixing and holding the adjusting knob 22a.

With the focus adjusted in this way, C
An image of the sample is picked up by the CD camera 8, and the signal thereof is taken in and stored in the control computer 9. Next, a control signal is output from the control computer 9 to the piezoelectric element driver 10 to control the piezoelectric element driver 10 to slightly expand and contract, and the one-dimensional lattice 3 connected to the piezoelectric element 21 is converted into a lattice. Direction of arrangement (direction of arrow A)
Move to. At this time, the expansion / contraction length of the piezoelectric element 21 is set to 1/3 of the lattice pitch of the one-dimensional lattice 3. Then, the lattice pattern (stripes) illuminated on the sample on the sample mounting surface 15a has a phase shifted by 1/3. Then, the sample image at this time is picked up by the CCD camera 8 in the same manner as described above, and the signal is taken by the control computer 9
It is taken in and memorized.

Similarly, the piezoelectric element 21 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 by the equation (3) using I ₁ , I ₂ , and I ₃ in the above equation (3) as the images picked up in the above manner, then the 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 12, and an image at a focused height is captured in the same manner as above. Sample stage moving mechanism 1 at this time
2 to input the movement information by 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.

When the axial chromatic aberration in the objective lens 6 caused by the difference between the excitation light wavelength and the fluorescence wavelength is corrected by operating the adjustment knob 22a in the position adjusting device 20, the adjustment position of the adjustment knob 22a is set to the objective lens. It is uniquely determined according to the six types, the excitation light and the wavelength of the fluorescence. For this reason, if the type of the objective lens 6 and the adjustment position of the adjustment knob 22a for the wavelengths of the excitation light and the fluorescence are measured or set in advance and a mark is provided at this adjustment position, this adjustment work will be performed. It will be very easy. In general, there are various combinations of the excitation light wavelength and the fluorescence wavelength when performing fluorescence microscope observation, but in the application of observing with a specific fluorescent probe on a biological sample, the combination of excitation light and fluorescence is This is almost fixed, and if a mark is provided at the adjustment position of the adjustment knob 22a for these, this observation becomes easy.

Next, a different embodiment of the grating illumination microscope according to the present invention will be described with reference to FIG. In addition,
In this embodiment, the same components as those of the device shown in FIG. 1 are designated by the same reference numerals, and the description thereof will be omitted. A different structure will be mainly described.

This device (the device shown in FIG. 2) is constructed by only having the piezoelectric element 18 in place of the position adjusting device 20 in the device shown in FIG. Therefore, in this apparatus, the piezoelectric element can be expanded and contracted by the piezoelectric element driver 10 to move the one-dimensional grating 3 in the direction of arrow A, but as in the apparatus of FIG. It cannot be moved. However, this apparatus is provided with the correction optical element 30 after the illumination lens 4, and the correction optical element 30 corrects the focal position shift caused by the axial chromatic aberration described above.

That is, in this apparatus, as described above, the axial chromatic aberration in the objective lens 6 due to the difference in the wavelengths of the excitation light and the fluorescence is corrected by the correction optical element 30, so that it is placed on the sample. Excitation light having a lattice pattern can be irradiated in a focused state, and the CCD camera 8 in a state where the lattice pattern image is clearly superimposed on the sample image.
The image of the sample can be captured by. Therefore, this image pickup signal is fetched and stored in the control computer 9. Thereafter, similarly to the apparatus shown in FIG. 1, the piezoelectric element 18 moves the one-dimensional grating 3 by 1/3 and 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. And the signal is sent to the control computer 9
It is taken in and memorized.

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, and the focus on the sample is calculated. Only get images at matching heights. And the sample table 15
Is moved in the optical axis direction by the sample table moving mechanism 12 and an image at a focused height is captured in the same manner as above. The movement information by the specimen table moving mechanism 12 at this time is input to the control computer 9, and the movement information and the image obtained as described above are combined and image-processed, so that the specimen mounting surface 15a is subjected to image processing. It is possible to obtain a three-dimensional image of the mounted sample.

In this device, the axial chromatic aberration generated in the objective lens 6 due to the difference in wavelength between the excitation light and the fluorescence is corrected by the correction optical element 30 arranged in the illumination optical system. As shown by the broken line in FIG. 2, a correction optical element 31 may be provided in the imaging optical system to correct this axial chromatic aberration.

Alternatively, the micrometer mechanism 32 having the same structure as the micrometer mechanism 22 used in FIG. 1 may be arranged to adjust the position of the image sensor 8 to correct the axial chromatic aberration.

[0054]

As described above, according to the present invention,
Since the grating illumination microscope has focus correction means for adjusting the focus of at least one of the illumination optical system and the imaging optical system in accordance with the wavelengths of the illumination light and the observation light, for example, a normal white light source as the illumination light. When using a general grating illumination microscope that forms and observes the reflected light from the sample using the fluorescence microscope as it is, the wavelength of the excitation light used as the illumination light by the focus correction means and the fluorescence emitted from the sample If the focus position is adjusted according to the difference with the wavelength, an ordinary grating illumination microscope can be easily used for fluorescence microscope observation. Furthermore, when performing fluorescence microscope observation, even if the wavelength of the fluorescence emitted using different excitation light of different wavelengths, just by adjusting the focus position corresponding to the wavelength of these excitation light and fluorescence by the focus correction means, You can easily respond.

[Brief description of drawings]

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

FIG. 2 is a schematic diagram illustrating a configuration of a grating illumination microscope according to a second embodiment of the present invention.

FIG. 3 is a schematic diagram illustrating a configuration of a conventional grating illumination microscope.

[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 15 specimen table 18,21 Piezoelectric element 20, 32 Position adjustment device 22 micrometer mechanism 22a Adjustment knob 30, 31 Correction optical element

Claims (12)

[Claims] 1. An illumination light source that emits illumination light, a sample table on which a sample is placed, an objective lens disposed above the sample table, and an illumination light beam emitted from the illumination light source. An illumination optical system that illuminates a sample on the sample table through an objective lens in a lattice pattern, and the sample that receives observation light emitted from the sample on the sample table illuminated by the lattice pattern through the objective lens An image forming optical system for forming an image of, the illumination light and the observation light have different wavelengths, the focus of the illumination optical system at the wavelength of the illumination light is matched, and the wavelength of the observation light. 2. A grating illumination microscope, comprising: focus correction means for adjusting the focus of at least one of the illumination optical system and the image forming optical system so that the focus of the image forming optical system in FIG. 2. A sample that receives excitation light and emits fluorescence is placed on the sample table, the excitation light is used as the illumination light, and the observation light is fluorescence emitted from the sample. The grating illumination microscope according to claim 1. 3. The focus position adjustment of the illumination optical system so that the focus correction means focuses the illumination of the lattice pattern when the illumination optical system illuminates the sample on the sample table with the lattice pattern. The method according to claim 1, wherein
The lattice illumination microscope according to any one of to 2. 4. The lattice pattern, wherein the illumination optical system has a transmittance distribution corresponding to the lattice pattern, and a grating member for converting transmitted light into illumination light of the lattice pattern and emitting the light, and the objective lens. And an illumination lens for projecting the image of the lattice pattern onto a sample placed on the sample table, the focus correction means adjusting the position of the lattice member in the optical axis direction. 4. The grating illumination microscope according to claim 3, comprising grating position adjusting means. 5. The grid position adjusting means includes a grid moving mechanism that moves the grid member in the axial direction, and a grid operating member that is externally operated to operate the grid moving mechanism. The grating illumination microscope according to claim 4, wherein a mark indicating an operation position corresponding to a difference in wavelength between the illumination light and the observation light is provided. 6. The correction optical element for an illumination optical system arranged in the optical path of the illumination optical system to adjust the focus position of the illumination optical system, wherein the focus correction means comprises a correction optical element. The grating illumination microscope according to. 7. The correction optical element for the illumination optical system is detachable and replaceable, and the types of the illumination light and the observation light,
The grating illumination microscope according to claim 6, wherein the grating illumination microscope is exchangeable according to the type of the objective lens. 8. The image forming means for focusing the image when the focus correcting means forms an image of the sample by receiving the observation light emitted from the sample on the sample table by the image forming optical system. 3. The grating illumination microscope according to claim 1, wherein the focal position of the optical system is adjusted. 9. The image forming optical system correction optical element is provided in the optical path of the image forming optical system to adjust the focus position of the image forming optical system. The grating illumination microscope according to claim 8. 10. The correction optical element for the imaging optical system is detachable and replaceable, and is replaced according to the types of the illumination light and the observation light and the type of the objective lens. The grating illumination microscope according to claim 9, which is characterized in that. 11. An image pickup device for picking up an image of the specimen formed by the image forming optical system, wherein the focus correction means adjusts a position of the image pickup device in an optical axis direction. The grating illumination microscope according to claim 8, wherein 12. The image pickup device position adjusting means comprises an image pickup device moving mechanism for moving the image pickup device in an axial direction, and an image pickup device operating member for externally operating the image pickup device moving mechanism. The grating illumination microscope according to claim 11, wherein the element operating member is provided with a mark indicating an operation position corresponding to a difference in wavelength between the illumination light and the observation light.