JP2005062515A - Fluorescence microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence microscope whose focus position can precisely be detected without exerting any influence on an observation of a fluorescent image. <P>SOLUTION: A dichroic mirror 17 for introducing infrared illumination light for automatic focusing is arranged in the optical path between a dichroic mirror 23 for introducing illumination light L1 and an objective 8. In automatic focusing, the infrared illumination light generated by an automatic focusing device 2 is reflected by the dichroic mirror 17 to light up a sample 5 and reflected infrared light from the sample is reflected by the dichroic mirror 17 and detected. In fluorescent observation, the sample 5 is irradiated with the illumination light L1 introduced by the dichroic mirror 23 transmitted through the dichroic mirror 17 and fluorescent light L2 from the sample 5 is transmitted through the dichroic mirror 17 and imaged by an imaging lens 6. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a fluorescence microscope equipped with a focal position detection device.

Conventionally, as a method of detecting the focus of a microscope, a contrast method in which the contrast of a photographed image is analyzed and the place where the contrast is the highest is a focus position, or light for focus detection is irradiated to the sample through an objective lens, A method of detecting the focal point by detecting the return light is known.
In the latter method, infrared light may be used as a light source for focus detection. Infrared light for focus detection is introduced into the microscope, and the sample is illuminated through the objective lens. The light reflected in the sample is collected by the objective lens, passes through the same optical path, and is detected by the photodetector for focus detection (see, for example, Patent Document 1).

Japanese Patent Laid-Open No. 13-242375

However, in the case of a fluorescence microscope that detects fluorescence, focus detection is performed with a focus detection device using infrared light, and the sample is excited with epi-illumination light to measure fluorescence. In this case, a focus detection mirror such as a half mirror that reflects infrared light into the optical path, enters the objective lens, irradiates the sample, and directs the reflected light from the sample to the detector, and fluorescence excitation light. A fluorescent mirror such as a half mirror that reflects and transmits fluorescence is inserted into the optical path. Therefore, the fluorescence may be reduced by the focus detection mirror, or the focus detection infrared light may be weakened by the fluorescence mirror, and the focus may not be detected.
Accordingly, an object of the present invention is to provide a fluorescence microscope capable of detecting a focal position with high accuracy without affecting the observation of a fluorescence image.

According to a first aspect of the present invention, a fluorescence microscope irradiates a sample with a focus position detecting light beam having a wavelength different from the wavelength of the observation light beam, and detects the focus position of the objective lens based on the reflected light from the sample. Is provided. And disposed in the optical path of the observation beam between the objective lens and the dichroic mirror for fluorescence observation that reflects the illumination light from the illumination optical system to the objective lens, and transmits the observation beam, and the focal position An optical member is provided that reflects the focus position detection light beam from the detection device to the sample and reflects the focus position detection light beam reflected by the sample to the focus position detection device.
A second aspect of the present invention is the fluorescent microscope according to the first aspect, wherein the optical member transmits light in a specific wavelength region including the observation light beam, and includes a focal position detection light beam and is different from the specific wavelength region. This is a dichroic mirror that reflects the light.
According to a third aspect of the present invention, in the fluorescence microscope according to the second aspect, the transmission wavelength range of the optical member includes the transmission wavelength range of the dichroic mirror for fluorescence observation and is wider than the transmission wavelength range.

  The present invention can provide a fluorescence microscope that can accurately detect a focal position without affecting the observation of a fluorescence image.

  Hereinafter, the best mode for carrying out the present invention will be described with reference to the drawings. FIG. 1 is a diagram showing a schematic configuration of an upright fluorescent microscope. The microscope includes an epi-illumination device 1 for observation, a slit projection type autofocus device 2 and a drive control system 3 for moving the stage 4 up and down. The specimen to be observed is sandwiched between the slide glass 5b and the cover glass 5a, and these samples 5 are placed on the stage 4.

  When performing fluorescence observation, the sample 5 is illuminated by the observation illumination device 1. A mercury lamp or the like is used as the light source 21 of the observation illumination device 1. The light beam emitted from the light source 21 is imaged near the pupil position of the objective lens 8 by the illumination optical systems 22a and 22b. The light beams emitted from the illumination optical systems 22 a and 22 b are incident on the excitation filter 24 provided in the filter unit 26. The excitation filter 24 is a filter that transmits a light beam in a wavelength region that excites a fluorescent substance in a specimen.

  The illumination light beam L1 that has passed through the excitation filter 24 is reflected by the dichroic mirror 23 for fluorescence observation, passes through the infrared cut filter 19 and the dichroic mirror 23 for autofocus, and is irradiated onto the sample 5 through the objective lens 8. . Although details will be described later, the infrared cut filter 19 and the dichroic mirror 23 are set so as to transmit light in the wavelength region of the fluorescent light L2 emitted from the illumination light beam L1 and the specimen.

  When the illumination light beam L1 transmitted through the excitation filter 24 is irradiated onto the specimen of the sample 5, the fluorescent material contained in the specimen is excited to generate fluorescence L2. The wavelength of the fluorescence L2 is shifted to a longer wavelength side than the wavelength of the illuminated illumination light beam L1. The fluorescence L2 passes through the objective lens 8 and becomes a parallel light beam, passes through the dichroic mirror 17 and the infrared cut filter 19, and enters the dichroic mirror 23.

  Since the fluorescence L2 is shifted to the longer wavelength side than the illumination light beam L1 as described above, it passes through the dichroic mirror 23. The absorption filter 25 absorbs light in a wavelength region other than the fluorescence L2 used for observation. The fluorescence L2 that has passed through the absorption filter 25 is imaged at the imaging position 7 by the imaging lens 6. The excitation filter 24, the dichroic mirror 23, and the absorption filter 25 are all provided in one filter unit 26, and these constitute a filter set corresponding to one type of excitation light. The observation illumination device 1 can be used by replacing the filter set according to the fluorescent material contained in the specimen.

  FIG. 2 is a configuration diagram similar to FIG. 1, but shows a light beam L3 for explaining the autofocus device 2 in place of the illumination light beam L1 and the fluorescence L2. As the light source 11, an LED or the like that generates near-infrared light is used. In front of the light source 11, a slit plate 12 having a horizontally long rectangular slit formed in a direction perpendicular to the paper surface is disposed. When the coordinate axis is set as shown in FIG. 2, a rectangular slit whose long side is parallel to the y-axis and whose short side is parallel to the z-axis is formed in the slit plate 12.

  The infrared illumination light L3 generated by the light source 11 is converted into a slit-shaped light beam by the projection lens 13 after passing through the slit plate 12. In this infrared illumination light L3, the light beam corresponding to the upper half side of the cross section is shielded by the pupil limiting diaphragm 16A, and the lower half of the slit light beam is incident on the half mirror 15. Part of the infrared illumination light L3 incident on the half mirror 15 passes through the half mirror 15, and further passes through the filter 18 that cuts visible light.

  By providing this filter 18, the incidence of visible light to the autofocus device 2 is blocked, and malfunctions during autofocus are prevented. Further, by providing the infrared cut filter 19 between the dichroic mirror 17 and the dichroic mirror 23, leakage of the infrared illumination light L3 toward the imaging lens 6 can be prevented.

  The infrared illumination light L3 that has passed through the filter 18 is incident on a focusing dichroic mirror 17 inserted between the fluorescence observation dichroic mirror 23 and the objective lens 8. The dichroic mirror 17 has a characteristic of transmitting the excitation light L1 and the fluorescence L2 (see FIG. 1) and reflecting the infrared illumination light L3. The infrared illumination light L3 that has passed through the filter 18 is reflected by the dichroic mirror 17 toward the objective lens 8 and applied to the sample 5.

  The infrared illumination light L3 irradiated on the sample 5 is reflected on the surface of the cover glass 5a, the interface between the cover glass 5a and the specimen, or the like. The reflected infrared light L 4 passes through the objective lens 8 and is reflected by the dichroic mirror 17. The reflected infrared light L4 reflected by the dichroic mirror 17 passes through the filter 18, and a part of the reflected infrared light L4 is reflected by the half mirror 15 in the z-axis direction. The reflected infrared light L4 reflected by the half mirror 15 is imaged by the imaging lens 14 after passing through the pupil limiting diaphragm 16B, and a slit image is formed on the photoelectric conversion element 20.

  The area shielded by the pupil limit stop 16B corresponds to the area shielded by the pupil limit stop 16A. Further, as the photoelectric conversion element 20, for example, a CCD imaging element is used, and a line sensor in which light receiving elements are arranged in a line shape may be used, or an area sensor in which light receiving elements are arranged two-dimensionally. good.

  The output signal from the photoelectric conversion element 20 is input to the signal processing unit 31, and the focal position is calculated. When the position of the sample 5 is shifted in the vertical direction (z direction) from the in-focus position, the image position of the slit image on the photoelectric conversion element 20 is also shifted in the x direction according to the shift amount. That is, the signal processing unit 31 calculates a focal position that is a relative distance from the in-focus position of the objective lens 8 according to the x-direction deviation amount of the imaging position.

  The drive unit 32 of the drive control system 3 moves the stage 4 up and down in the z direction. The CPU 41 controls the driving unit 32 based on the focus information input from the signal processing unit 31 and moves the stage 4 to the in-focus position. The memory 42 stores various parameters related to autofocus. The input unit 43 is provided with a switch for instructing autofocus, a switch for manually moving the stage 4 up and down, and the like.

  Next, with reference to FIG. 3, the setting of the wavelength region of the dichroic mirror 17 will be described. In FIG. 3, the horizontal axis represents the wavelength of light, and the vertical axis represents the transmittance of each optical member. A curve 53 shows the transmission characteristics of the excitation filter 24, and transmits light (blue) in the vicinity of a wavelength of 500 nm as excitation light (illumination light beam L1). A curve 54 represents the transmission characteristics of the absorption filter 25. The transmittance is large in a wavelength region centered at about 550 nm shifted to the longer wavelength side than the excitation filter 24. The transmission wavelength range of the absorption filter 25 is set so as to include the wavelength range of the fluorescence L2.

  A curve 52 shows the transmission characteristics of the dichroic mirror 23, and the transmittance is almost 0% in the transmission wavelength region of the excitation filter 24. When the wavelength is slightly larger than the transmission wavelength region of the excitation filter 24, the transmittance increases rapidly, and a large state is maintained up to a wavelength slightly smaller than 700 nm. That is, the dichroic mirror 23 reflects the light that has passed through the excitation filter and transmits light in a wavelength region that includes the fluorescence L2.

  On the other hand, the transmission characteristic of the dichroic mirror 17 for autofocusing is such that the visible region is transmitted from the near ultraviolet region and the near infrared region is reflected as indicated by a curve 51 so as not to affect the fluorescence observation. Are set to various characteristics. That is, the transmittance is set to be very small at about 0% on the long wavelength side including the wavelength region of the infrared illumination light L3 with about 700 nm as a boundary, and the transmittance is set to about 100% on the short wavelength side. The

  Specifically, it is desirable that the transmittance at a wavelength of 360 nm to 650 nm is 70% or more and the transmittance at a wavelength of 750 nm to 850 nm is 10% or less. Furthermore, it is more desirable if the transmittance at a wavelength of 400 nm to 650 nm is 85% or more and the transmittance at a wavelength of 750 nm to 850 nm is 5% or less.

  When the transmission characteristics of the dichroic mirror 17 are set as described above, the illumination light beam L1 reflected by the dichroic mirror 23 and the fluorescence L2 from the sample 5 are transmitted through the dichroic mirror 17. Further, the infrared illumination light L3 for autofocus is reflected by the dichroic mirror 17 and does not reach the dichroic mirror 23 side. Therefore, the presence of the dichroic mirror 17 does not affect the fluorescence observation, and it is possible to always perform an autofocus operation separately from the fluorescence observation. Furthermore, since near-infrared light reflected by the dichroic mirror 17 is used for autofocus illumination, it is possible to avoid the fading of fluorescence that occurs when focus adjustment is performed using fluorescence.

  The filter unit 26 in FIG. 1 is replaced with an optimum unit according to the fluorescent material, but the transmission wavelength ranges of the excitation filter 24, the dichroic mirror 23, and the absorption filter 25 provided in the filter unit 26 are as shown in FIG. As long as it is included in the transmission wavelength range of the dichroic mirror 17, it can be used for fluorescence observation.

  In the above-described embodiment, the upright fluorescent microscope has been described as an example. However, the present invention can also be applied to an inverted fluorescent microscope. FIG. 4 is a schematic diagram showing a schematic configuration of an inverted fluorescence microscope. An opening 4 a is formed in the stage 4 on which the sample 5 is placed, and the objective lens 8 is provided below the stage 4. In the inverted fluorescence microscope, the specimen is observed from below the sample 5 through the opening 4a.

  An autofocus device 2 is provided below the objective lens 3, and an observation illumination device 1 is further provided below the autofocus device 2. That is, the autofocus dichroic mirror 17 is arranged on the observation optical path between the dichroic mirror 23 for fluorescent illumination and the objective lens 8. The observation illumination device 1 and the autofocus device 2 are the same as those shown in FIGS. The drive control system 3 drives the objective lens 8 and moves the objective lens 8 up and down.

  The illumination light beam (excitation light) generated by the light source 21 and transmitted through the excitation filter 24 is reflected upward (z direction) by the fluorescence observation dichroic mirror 23 and transmitted through the infrared cut filter 19 and the autofocus dichroic mirror 17. After that, the sample 5 is illuminated from the bottom side through the objective lens 8. Fluorescence generated from the sample 5 passes through the objective lens 8 to become a parallel light beam, passes through the dichroic mirror 17, the infrared cut filter 19, and the dichroic mirror 23, and forms an intermediate image at a position 49 by the relay lens 47a and the reflection mirror 48a. Is done. Further, an observation image is formed at the position 7 by the relay lens 47b, the reflection mirror 48b, and the imaging lens 6.

  On the other hand, the infrared illumination light for autofocus is reflected by the dichroic mirror 17 disposed on the observation optical path, and is irradiated onto the sample 5 through the objective lens 8. The infrared illumination light reflected by the sample 5 passes through the objective lens 8, is reflected by the dichroic mirror 17, and is taken into the autofocus device 2 side. In the case of the apparatus shown in FIG. 4 as well, the transmission characteristics of the dichroic mirror 17 are set as shown by the curve 51 in FIG. There is no effect.

  FIG. 5 shows a modification of the above-described fluorescence microscope, and a light shielding filter 60 is added to the autofocus device 2. By the way, as shown in FIG. 6, the slit-shaped infrared illumination light L3 may be partially reflected at the central portion of the objective lens 8. Depending on the type of the objective lens 8, a material having a very high reflectance may be used. In such a case, the light reflected by the central portion of the objective lens 8 is also detected by the photoelectric conversion element 20. As a result, the focus position may not be normally detected.

  However, in the autofocus device 2 shown in FIG. 5, such an adverse effect can be prevented by shielding the light beam at the central portion using the light shielding filter 60. FIG. 7A is a diagram showing the light shielding filter 60, and a light shielding region 60b is formed at the center of the transmission region 60a. Therefore, as shown in FIG. 7B, the slit-like infrared illumination light 61 is shielded at the upper half by the pupil limiting diaphragm 16 </ b> A, and further, the central portion is shielded by the light shielding part 60 b of the light shielding filter 60.

  When the light shielding filter 60 is used, since the infrared illumination light L3 other than the hatched portion is incident on the sample 5 as shown in FIG. 8, the light flux near the optical axis is not incident on the objective lens 8. As a result, the generation of the light L0 reflected from the central portion of the objective lens 8 shown in FIG. 6 can be prevented, and the focal position detection operation of the autofocus device 2 is not affected.

  Instead of inserting the light shielding filter 60 as shown in FIG. 5, the shape of the pupil limiting diaphragm 16A may be made as shown in FIG. 9A so that the light shielding filter 60 is also used. That is, the semicircular light shielding portion 160 is formed at a position corresponding to the optical axis of the pupil limiting diaphragm 16A. As a result, as shown in FIG. 9B, the central portion of the slit-shaped infrared illumination light 61 is shielded by the light shielding portion 160.

  In the correspondence between the embodiment described above and the elements of the claims, the illumination light beam L1 and the fluorescence L2 are the observation light beam, the infrared illumination light L3 and the reflected infrared light L4 are the focus position detection light beam, The focus device 2 constitutes a focal position detection device, the observation illumination device 1 constitutes an illumination optical system, the dichroic mirror 23 constitutes a fluorescence observation dichroic mirror, and the dichroic mirror 17 constitutes an optical member. In addition, the present invention is not limited to the above embodiment as long as the characteristics of the present invention are not impaired.

It is a schematic diagram which shows schematic structure of an erecting fluorescence microscope. It is a figure explaining the autofocus device. It is a figure which shows wavelength ranges, such as a dichroic mirror. It is a schematic diagram which shows schematic structure of an inverted fluorescence microscope. It is a figure which shows the modification of the fluorescence microscope shown in FIG. FIG. 6 is a diagram showing an optical path of slit-shaped illumination light that enters a sample 5. It is a figure explaining the light shielding filter 60, (a) The top view of the light shielding filter 60, (b) is a figure which shows the cross-sectional shape of the slit-shaped infrared illumination light 61 when the light shielding filter 60 is used. It is a figure showing the state of the slit illumination light which injects into the sample 5 when the optical axis vicinity of slit illumination light is shielded. It is a figure which shows the modification of the pupil limiting aperture stop 16A.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Observation illumination apparatus 2 Autofocus apparatus 3 Drive control system 4 Stage 5 Sample 6 Imaging lens 8 Objective lens 16A, 16B Pupil limiting diaphragm 17, 23 Dichroic mirror 18 Visible cut filter 19 Infrared cut filter 24 Excitation filter 25 Absorption filter 26 Filter unit 60 Light blocking filter L1 Illumination beam L2 Fluorescence L3 Infrared illumination light L4 Reflected infrared light

Claims (3)

A fluorescence microscope including a focal position detection device that irradiates a sample with a focal position detection light beam having a wavelength different from the wavelength of the observation light beam, and detects a focal position of an objective lens based on reflected light from the sample,
Arranged in the optical path of the observation light beam between the objective lens and the illumination dichroic mirror that reflects the illumination light from the illumination optical system to the objective lens, and transmits the observation light beam, and the focus An optical member is provided that reflects the focal position detection light beam from the position detection device to the sample and reflects the focal position detection light beam reflected by the sample to the focus position detection device. Fluorescence microscope. In the fluorescence microscope according to claim 1,
The optical member is a dichroic mirror that transmits light in a specific wavelength region including the observation light beam and reflects light in a wavelength region different from the specific wavelength region including the focal position detection light beam. Fluorescence microscope. The fluorescence microscope according to claim 2,
The fluorescence microscope characterized in that a transmission wavelength range of the optical member includes a transmission wavelength range of the fluorescence observation dichroic mirror and is wider than the transmission wavelength range.

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