JP2004138819A - Confocal fluorescence microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal fluorescence microscope capable of always securing a bright fluorescent image and realizing high-quality fluorescence observation. <P>SOLUTION: A rotary disk 23 provided with a pattern area 231 provided with a light transmission part having characteristic that the wavelength of light to excite fluorescence is transmitted, and a pattern area 232 provided with a light transmission part having characteristic that the wavelength of the fluorescence is transmitted is rotated by a motor 34. Then, light from a light source 21 is made incident on the light transmission part of the pattern area 231, a sample 29 is irradiated with the light transmitted through the light transmission part, and the fluorescence generated from the sample 29 is formed into an image at the light transmission part of the pattern area 232. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a fluorescence microscope for observing a fluorescence of a biological specimen by enlarging the fluorescence, and more particularly to a fluorescence confocal microscope for observing a three-dimensional structure and a high contrast of the fluorescence specimen.
[0002]
[Prior art]
Recently, genetic research has become increasingly popular, and fluorescence observation has been used for genetic analysis and the like in this genetic research.
[0003]
In such fluorescence observation, a fluorescence confocal microscope using a sectioning effect of a confocal method has been known as one that observes fluorescence with good contrast.
[0004]
As the fluorescent confocal microscope, a disk scan type confocal microscope is used in which a pattern having a large number of minute apertures is formed on a disk, and the disk is rotated and scanned to obtain a confocal image. Also, a well-known Nipkow disk using a pinhole pattern is the most frequently used disk.
[0005]
FIG. 6 shows an example of a fluorescence confocal microscope using a Nippow disk, in which a condenser lens 2 and a dichroic mirror 3 are arranged on the optical path of light emitted from a light source 1 such as a mercury light source. The dichroic mirror 3 reflects a wavelength that excites fluorescence and transmits a fluorescence wavelength. A sample 7 is placed on the reflection optical path of the dichroic mirror 3 via a Nipkow disk 4, a first imaging lens 5, and an objective lens 6.
[0006]
Here, the Nippow disk 4 has a spiral arrangement of the pinholes 4a as shown in FIG. 7, and the distance between the pinholes 4a is about 10 times the diameter of the pinhole 4a. . The Nippow disk 4 is connected to a motor shaft (not shown) via a rotation shaft 8 so as to rotate at a constant rotation speed.
[0007]
A CCD camera 10 is arranged on a transmission optical path of the dichroic mirror 3 with respect to the sample 7 via a second imaging lens 9. The image output terminal of the CCD camera 10 is connected to a monitor 11, and displays an image captured by the CCD camera 10.
[0008]
In such a configuration, light emitted from the light source 1 passes through the condenser lens 2, and only a short wavelength serving as excitation light is reflected by the dichroic mirror 3 and is incident on the Nippow disk 4 rotating at a constant speed. The light passing through the pinhole 4a of the Nippow disk 4 passes through the first imaging lens 5, is imaged by the objective lens 6, and is incident on the sample 7.
[0009]
In the sample 7, fluorescence is generated by the incident excitation light. The fluorescence from the sample 7 is projected through the objective lens 6 by the first imaging lens 5 onto the Nippow disk 4. The focused portion of the projected sample image passes through the pinhole 4a, and a long wavelength, which is fluorescent light, is transmitted by the dichroic mirror 3 in the direction of the second imaging lens 9, and the second imaging lens 9 transmits the second image. An image is captured by a CCD camera 10 via an imaging lens 9. The confocal image captured by the CCD camera 10 is displayed on a monitor 11. In this case, although not shown, visual observation may be performed using an eyepiece instead of the second imaging lens 9 and the CCD camera 10.
[0010]
According to such a fluorescence confocal microscope, since only an in-focus image passing through the pinhole 4a of the Nippow disk 4 can be observed, the sample can be observed by moving the focus up and down (Z direction). It is possible to observe an image for each height of 7 so-called sectioning images.
[0011]
However, when performing fluorescence observation with a confocal microscope using such a Nipkow disk 4, the total area of the pinholes 4a of the Nipkow disk 4 is about 1/100 of the entire disk, and most of the light from the light source 1 Does not pass through the disc, a very dark image is observed. This means that, in particular, the fluorescence is extremely weak at 1 / 100,000 or less with respect to the excitation light, and therefore, it becomes very difficult to observe high-quality fluorescence with a confocal microscope using the Nippow disk 4.
[0012]
Therefore, conventionally, a disk scanning type microscope using a microlens array disclosed in Japanese Patent Application Laid-Open No. 2000-206412 has been considered as a disk scanning type that enables brighter fluorescence observation.
[0013]
FIG. 8 shows a schematic configuration of a disk scanning microscope provided with such a microlens. In this case, a microlens plate 12 having a large number of microlenses and a pinhole plate 13 having pinholes arranged at the same position as the microlenses of the microlens plate 12 are provided, and the laser light emitted from the laser light source is provided. Numeral 14 focuses on the pinholes of the pinhole plate 13 via the microlenses of the microlens plate 12. Here, the pinholes of the pinhole plate 13 are the same as those of the above-described Nippow disk 4, and are as shown in FIG. The microlens plate 12 and the pinhole plate 13 are connected to a motor shaft (not shown) via a rotation shaft 15 so that the microlens plate 12 and the pinhole plate 13 rotate at a constant rotation speed as shown by an arrow.
[0014]
A dichroic mirror 16 is arranged between the microlens plate 12 and the pinhole plate 13. The dichroic mirror 16 transmits the wavelength of the laser light and reflects the fluorescence wavelength. A sample 18 is disposed on a transmission optical path of the dichroic mirror 16 via an objective lens 17. A CCD camera 20 is arranged on the reflected light path of the dichroic mirror 16 via an eyepiece 19.
[0015]
In such a configuration, the laser light 14 emitted from the laser light source enters the microlens plate 12, passes through the respective microlenses on the microlens plate 12, passes through the dichroic mirror 16, and passes through the pinhole plate. The light is focused on each of the 13 pinholes. The light passing through the pinhole of the pinhole plate 13 forms an image by the objective lens 17 and enters the sample 18.
[0016]
In this state, the microlens plate 12 and the pinhole plate 13 perform scanning by rotating around the rotation shaft 15 by rotation of the rotation shaft 15.
[0017]
In the sample 18, fluorescence is generated by the incident excitation light. The fluorescence from the sample 18 projects an image of the sample on the pinhole plate 13 via the objective lens 17. An in-focus portion of the projected sample image passes through the pinhole, and a long wavelength, which is fluorescence, is reflected by the dichroic mirror 16 in the direction of the eyepiece 19 and passes through the eyepiece 19 through the CCD. The image is taken by the camera 20.
[0018]
[Problems to be solved by the invention]
However, even with such a configuration, in addition to the pinhole plate 13 for confocal observation, as well as a dichroic mirror 16 for fluorescence observation, an excitation filter and an absorption filter (not shown) are required. As described above, when the very weak fluorescent light emitted from the sample 18 passes through the pinhole plate 13, the dichroic mirror 16, and the absorption filter, it is further attenuated by a loss of light amount generated by passing through the pinhole plate 13, the dichroic mirror 16, and the bright fluorescent light. There is a problem that it is difficult to obtain an image, and high-quality fluorescence observation cannot be performed.
[0019]
The present invention has been made in view of the above circumstances, and has as its object to provide a fluorescence confocal microscope capable of securing a bright fluorescent image and realizing high-quality fluorescence observation.
[0020]
[Means for Solving the Problems]
According to the first aspect of the present invention, a light source, a first pattern region and a second pattern region having a light transmitting portion and a light blocking portion are formed, and the light transmitting portion of the first pattern region is formed of the light source. The second pattern region has a characteristic of transmitting light of a wavelength selected to excite fluorescence selected from light, and the light transmitting portion of the second pattern region includes a pattern region forming unit having a characteristic of transmitting the wavelength of fluorescence, and a light source. A first optical means for irradiating the sample with the light transmitted through the light transmitting portion of the first pattern and irradiating the sample with the light transmitted through the light transmitting portion; and a light transmitted through the light transmitting portion of the first pattern. Scanning means for scanning the sample with light, and second optical means for forming an image of fluorescence generated from the sample by the irradiated light on a light transmitting portion of the second pattern area. Features.
[0021]
According to a second aspect of the present invention, in the first aspect of the present invention, the pattern area forming means comprises a disk-shaped disk, and the scanning means comprises rotating means for rotating the disk.
[0022]
According to a third aspect of the present invention, in the second aspect of the present invention, the position detection means for detecting the position information of the first and second pattern areas of the pattern area forming means, and the light of the second pattern area. A third optical unit that forms an image of the fluorescence transmitted through the transmission unit, and an image formed by the third optical unit in synchronization with position information of the first and second pattern areas detected by the position detection unit And an imaging unit that captures the obtained fluorescence image.
[0023]
According to a fourth aspect of the present invention, in the first aspect of the present invention, the first and second pattern regions have slit-shaped light transmitting portions that transmit light arranged at equal intervals. It is characterized by.
[0024]
According to a fifth aspect of the present invention, in the first aspect of the present invention, the first and second pattern regions are formed of a pattern symmetrical with respect to a rotation axis of the disc. It is characterized by having.
[0025]
As a result, according to the present invention, the light from the light source is incident on the sample through the light transmission portion of the first pattern region having the property of transmitting the wavelength of the light that excites the fluorescence, and the fluorescence emitted from the sample is Since a confocal image can be obtained through the second pattern area light transmitting portion having the property of transmitting the wavelength of the fluorescent light, the functions of the excitation filter and the fluorescent filter, which are indispensable conventionally, are assigned to the common pattern area. The formation means (disk) can be provided, and the excitation filter and the fluorescence filter can be omitted.
[0026]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0027]
FIG. 1 shows a schematic configuration of a confocal microscope to which an embodiment of the present invention is applied.
[0028]
In this case, on the optical path of light emitted from a light source 21 such as a mercury light source, a collimator lens 22, a disc-shaped rotating disk 23 as a pattern area forming means, a relay lens 24 including a plurality of lenses, and a mirror 25 are arranged. Have been.
[0029]
A first imaging lens 26 and a dichroic mirror 27 are arranged on the reflection optical path of the mirror 25, and a sample 29 is arranged on the reflection optical path of the dichroic mirror 27 via the objective lens 28. I have.
[0030]
In this case, the collimator lens 22, the relay lens 24, the mirror 25, the first imaging lens 26, and the dichroic mirror 27 constitute a first optical system.
[0031]
On the transmission optical path of the dichroic mirror 27, a second imaging lens 30 and a rotating disk 23 constituting a second optical system are arranged, and further, a third imaging lens constituting a third optical system. A CCD camera 32 is provided as an image pickup means 31. In this case, the position of the rotating disk 23 on the transmitted light path of the dichroic mirror 27 is a position symmetrical with respect to the position of the rotating disk 23 on the light path on the light source side with respect to the rotation axis.
[0032]
Further, the rotating disk 23 is connected to a shaft of a motor 34 via a rotating shaft 23a as rotating means constituting a scanning means, and is rotated at a constant rotation speed.
[0033]
On an optical path between the third imaging lens 31 and the front surface of the CCD camera 32, a shutter 33 for turning ON / OFF light to the CCD camera 32 is arranged.
[0034]
A sensor 35 is disposed on the outer periphery of the rotating disk 23 as position detecting means for detecting position information of the pattern areas 231 and 232. The sensor 35 detects position information of the pattern areas 231 and 232 by reading a marker 235 described later on the rotating disk 23.
[0035]
A shutter control unit 36 is connected to the sensor 35. The shutter control unit 36 controls ON / OFF of the shutter 33 based on the output of the sensor 35 (presence or absence of the marker 235).
[0036]
FIG. 2A shows a schematic configuration of the rotary disk 23, in which pattern areas 231 and 232 in which slit-shaped light transmitting portions are arranged at equal intervals, and a fan-shaped light-shielding area which divides these pattern areas 231 and 232 are shown. 233 and 234 are formed. In this case, the pattern areas 231 and 232 are formed of patterns symmetric with respect to the center of the rotating disk 23, that is, the rotating shaft 23a. In addition, the rotary disk 23 is provided with a marker 235 that shields light over a half circumference along the circumferential direction of the periphery.
[0037]
In the pattern regions 231 and 232, slit-shaped light transmitting portions 236 through which light of a specific wavelength passes and light shielding portions 237 that block light are alternately arranged as shown in the enlarged view of FIG. Are located. In this case, the ratio of the width of the light transmitting portion 236 to the width of the light shielding portion 237 is set to, for example, 1: 9. The width W of the slit-shaped light transmission portion 236 through which light passes is M, the wavelength of light is λ, and the aperture ratio of the objective lens is NA, as in the case of the pinhole. Then, W = kλM / NA
Here, k is a coefficient, and k = 1 is often used.
[0038]
Further, these pattern regions 231 and 232 have different wavelengths that pass through the respective light transmitting portions 236. In the pattern region 231, as shown in FIG. 3A, the fluorescent light selected from the light of the light source 1 is excited. In this case, the pattern region 232 has a characteristic of transmitting the wavelength of the light to be transmitted and blocking other wavelengths (including the wavelength A of the fluorescent light). Has the characteristic of transmitting the wavelength A and blocking other wavelengths (wavelengths of light that excites fluorescence).
[0039]
The dichroic mirror 27 reflects the light having the wavelength of the light that excites the fluorescence transmitted through the pattern area 231 of the rotating disk 23 as shown in FIG. 3B, and from the sample 29 as shown in FIG. Has the characteristic of transmitting the wavelength A of the fluorescent light.
[0040]
Note that these wavelength characteristics differ depending on the fluorescent dye used. For example, when observing FITC, the excitation maximum wavelength is 490 nm and the fluorescence maximum wavelength is 520 nm. A wavelength of 460 to 490 nm is used for transmitting the dichroic mirror 27 and transmitting the dichroic mirror 27. The slit-shaped light transmitting part 236 of the pattern area 232 of the rotating disk 23 is used. 510 nm is used as the wavelength for transmitting light.
[0041]
Next, the operation of the embodiment configured as described above will be described.
[0042]
First, the motor 34 is driven to rotate the rotating disk 23 at a constant speed.
[0043]
In this state, the light emitted from the light source 21 passes through the collimator lens 22 and enters the rotating disk 23.
[0044]
Now, when light is incident on the pattern area 231 of the rotating disk 23, the light having a wavelength that excites fluorescence from the light from the light source 21 as shown in FIG. 3A by the slit-shaped light transmitting portion 236 of the pattern area 231. Are selectively transmitted, and other wavelengths are blocked.
[0045]
The light from the light source 21 whose wavelength has been selected passes through the relay lens 24, is reflected at a right angle by the mirror 25, and enters the dichroic mirror 27 through the first imaging lens 26.
[0046]
In this case, as shown in FIG. 3B, the dichroic mirror 27 reflects light having a wavelength of light that excites fluorescence transmitted through the pattern area 231 of the rotating disk 23, as shown in FIG. 3C. Since it has the property of transmitting the wavelength A of the fluorescence from the sample 29, the light that excites the fluorescence incident through the first imaging lens 26, that is, the excitation light is reflected, and the objective lens 28 And is incident on the sample 29.
[0047]
In the sample 29, fluorescence is generated by the incident excitation light. The fluorescence from the sample 29 passes through the objective lens 28, and then passes through the dichroic mirror 27, and forms an image of the sample 29 on the pattern area 232 on the rotating disk 23 via the second imaging lens 30. I do.
[0048]
As shown in FIG. 3C, the pattern region 232 has a characteristic of transmitting the wavelength A of the fluorescence from the sample 29 and blocking other wavelengths (the wavelength of the light for exciting the fluorescence). Accordingly, even if a part of the excitation light reflected and scattered by the sample 29 passes through the dichroic mirror 27 and enters the pattern region 232, the incident light is blocked and the focused fluorescent wavelength A Only the component passes through the slit-shaped light transmitting portion 236 and is imaged as a fluorescent image of the sample 29 on the CCD camera 32 via the third imaging lens 31.
[0049]
From this state, when the rotating disk 23 rotates and the pattern area 232 is located on the optical path of the light source 21 and the pattern area 231 is located on the photographing optical path of the CCD camera 32, the pattern area 232 is transmitted through the pattern area 232 of the rotating disk 23. Is only the fluorescence wavelength of the light from the light source 21 as shown in FIG. 3 (c), so that the fluorescence cannot be excited at the wavelength of the light incident on the sample 29 at this time.
[0050]
Then, when the rotating disk 23 is further rotated so that the pattern area 231 is positioned on the optical path of the light source 21 and the pattern area 232 is positioned on the shooting optical path of the CCD camera 32, the light source 21 is processed in the same manner as described above. The wavelength of the light that excites the fluorescence among the light from the sample passes through the pattern region 231 to excite the fluorescence of the sample 29, and the wavelength of the fluorescence from the sample 29 passes through the pattern region 232 and is imaged by the CCD camera 32. You.
[0051]
Such a state is detected by a positional relationship between the sensor 235 and the marker 235 provided over a half circumference along the circumferential direction of the peripheral portion of the rotating disk 23. That is, as shown in FIG. 4A, when the pattern area 231 is located on the optical path of the light source 21 and the pattern area 232 is located on the imaging optical path of the CCD camera 32, as shown in FIG. An OFF signal is output by shielding the light, and in a state where the pattern area 232 is located on the optical path of the light source 21 and the pattern area 231 is located on the imaging optical path of the CCD camera 32, the light shielding by the marker 235 is released. Outputs ON signal.
[0052]
When receiving the output signal from the sensor 35, the shutter controller 36 inverts the signal level as shown in FIG. When the pattern area 232 is positioned on the optical path of the CCD camera 32 on the optical path of the CCD camera 32, an OPEN signal for instructing the shutter to open is output, and the pattern area 232 is positioned on the optical path of the light source 21. When the 231 is located on the optical path for photographing of the CCD camera 32, a CLOSE signal for instructing to close the shutter is output.
[0053]
As a result, the period in which the shutter control unit 36 provides the OPEN signal for instructing the shutter to be opened to the shutter 33 positioned in front of the CCD camera 32, that is, the pattern area 231 of the rotating disk 23 on the optical path from the light source 21. However, only when each of the pattern regions 232 is located on the imaging optical path of the CCD camera 32, the wavelength of light that excites fluorescence among the light from the light source 21 passes through the pattern region 231 to excite the fluorescence of the sample 29, The wavelength of the fluorescence from the sample 29 passes through the pattern area 232 and is imaged by the CCD camera 32, so that a fluorescence image of the sample 29 is obtained.
[0054]
Therefore, according to this configuration, the functions of the excitation filter and the fluorescence filter, which are essential in the conventional fluorescence microscope, are changed to the pattern regions 231 and 232 in which the slit-shaped light transmitting portions formed on the rotating disk 23 are arranged at equal intervals. The excitation filter and the fluorescence filter can be omitted. This makes it possible to eliminate the loss of light amount caused by passing through these filters, so that a bright fluorescent image can be always obtained, and high-quality fluorescent observation can be performed.
[0055]
In the above-described embodiment, the ratio between the width of the light transmitting portion 236 and the width of the light shielding portion 237 in the pattern areas 231 and 232 of the rotating disk 23 is set to, for example, 1: 9. However, this ratio may be smaller or larger. For example, in the case of very dark fluorescent light, the out-of-focus component increases, but when the fluorescent light is set to about 1: 3, the fluorescent light image becomes bright. In the case of very bright fluorescent light, setting the ratio to 1:20 or 1:50 almost eliminates out-of-focus components and provides a sectioning image of only an in-focus image, but the fluorescent image in this case becomes darker. .
[0056]
In the embodiment described above, the pattern areas 231 and 232 of the rotating disk 23 have slit light transmitting portions arranged at equal intervals. However, the present invention can also be applied to the above-described Nippow disk having pinholes. it can. In this case, as shown in FIG. 5, two fan-shaped regions 501 and 502 having a pinhole pattern are arranged via light-shielding regions 503 and 504, and the pinhole portion of the region 501 is shown in FIG. In the same manner as the slit-shaped light transmitting portion of the patterned region 231, the excitation light wavelength of the light from the light source is transmitted, and the pinhole of the region 502 is formed by the slit-shaped slit of the patterned region 232 shown in FIG. If the wavelength of the fluorescent light from the sample is transmitted in the same manner as in the light transmitting portion, it can be used in the same manner as described above.
[0057]
In addition, the present invention is not limited to the above-described embodiment, and can be variously modified in an implementation stage without departing from the spirit of the invention.
[0058]
Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent features. For example, even if some components are deleted from all the components shown in the embodiments, the problem described in the column of the problem to be solved by the invention can be solved, and the problem described in the column of the effect of the invention can be solved. In the case where the effect described above is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0059]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a fluorescent confocal microscope capable of always securing a bright fluorescent image and realizing high-quality fluorescent observation.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a confocal microscope to which an embodiment of the present invention is applied.
FIG. 2 is a diagram showing a schematic configuration of a rotating disk used in the embodiment.
FIG. 3 is a diagram showing a pattern area of a rotating disk and characteristics of a dichroic mirror used in the embodiment.
FIG. 4 is a diagram for explaining operations of a sensor and a shutter control unit used in the embodiment;
FIG. 5 is a diagram showing a schematic configuration of another rotating disk used in the embodiment.
FIG. 6 is a diagram showing a schematic configuration of a conventional confocal microscope.
FIG. 7 is a diagram showing a schematic configuration of a Nippow disk used in a conventional confocal microscope.
FIG. 8 is a diagram showing a schematic configuration of a conventional disk scanning microscope provided with a microlens.
[Explanation of symbols]
21 ... Light source 22 ... Collimator lens 23 ... Rotating disk 23a ... Rotating axis 231.232 ... Pattern area 233.234 ... Shading area 235 ... Marker 236 ... Light transmitting part 237 ... Light shielding part 24 ... Relay lens 25 ... Mirror 26 ... No. 1 imaging lens 27 dichroic mirror 28 objective lens 29 sample 30 second imaging lens 31 third imaging lens 32 CCD camera 33 shutter 34 motor 35 sensor 36 shutter control Section 231 pattern area 231.232 pattern area 232 pattern area 233.234 light-shielding area 235 marker 236 light-transmitting section 237 light-shielding section 501.502 area 503.504 light-shielding area

Claims (5)

A light source,
A first pattern region and a second pattern region having a light transmitting portion and a light blocking portion are formed, and the light transmitting portion of the first pattern region is formed of light for exciting fluorescence selected from light of the light source. A light-transmitting portion of the second pattern region having a characteristic of transmitting a wavelength, a pattern region forming means having a characteristic of transmitting a wavelength of fluorescence,
First optical means for causing light from the light source to enter the light transmitting portion of the first pattern, and irradiating the sample with light transmitted through the light transmitting portion;
Scanning means for scanning the sample with light transmitted through the light transmitting portion of the first pattern;
A fluorescent optical confocal microscope, comprising: second optical means for forming an image of fluorescent light generated from the sample by the irradiated light on a light transmitting portion of the second pattern region. 2. The fluorescence confocal microscope according to claim 1, wherein said pattern area forming means comprises a disk-shaped disk, and said scanning means comprises a rotating means for rotating said disk. Position detecting means for detecting position information of the first and second pattern areas of the pattern area forming means, and third optical means for forming an image of fluorescence transmitted through a light transmitting portion of the second pattern area; And an image pickup means for picking up a fluorescent image formed by the third optical means in synchronization with position information of the first and second pattern areas detected by the position detection means. The fluorescence confocal microscope according to claim 2, wherein The fluorescence confocal microscope according to any one of claims 1 to 3, wherein the first and second pattern areas have slit-shaped light transmitting portions that transmit light arranged at equal intervals. The fluorescence confocal microscope according to any one of claims 2 to 4, wherein the first and second pattern regions have a pattern symmetrical with respect to a rotation axis of the disc.

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