JP2010026241A - Fluorescence microscope and focus detecting unit - Google Patents

Abstract

An object of the present invention is to perform focus detection with high accuracy regardless of the type of fluorescent reagent.
A fluorescence microscope apparatus of the present invention is for observation in which excitation light is irradiated to a fluorescent sample (0) through an objective lens (12), and fluorescence generated in the fluorescent sample is detected through the objective lens. An optical system (2) and a focus detection optical system (3) that irradiates the fluorescent sample with light for focus detection via the objective lens and detects the light reflected by the fluorescent sample via the objective lens. And a first switching means (201) for switching the focus detection wavelength, which is the wavelength of the light irradiated and detected by the focus detection optical system, between at least two different wavelengths.
[Selection] Figure 1

Description

  The present invention relates to a fluorescence microscope apparatus having a focus detection function of an objective lens, and a focus detection apparatus.

  Patent Document 1 discloses a slit projection type focus detection device mounted on a biological microscope. As shown in FIG. 1 of Patent Document 1, the slit-projection type focus detection apparatus uses a light emitted from an illuminated slit (reference numeral 22 of Patent Document 1) as one pupil of an objective lens (reference numeral 12 of Patent Document 1). Flood light only to the part. At this time, a slit image is formed on the specimen (reference numeral 18 in Patent Document 1), and the light reflected on the specimen passes through the other part of the pupil of the objective lens onto the CCD (reference numeral 30 in Patent Document 1). A slit image is formed. When the defocus amount of the objective lens (the positional relationship between the focal plane of the objective lens and the sample surface) changes in this state, the slit image formation position moves on the CCD. Therefore, the slit projection type focus detection device generates a defocus signal in accordance with the output signal of the CCD.

In this way, the focus detection device that irradiates the specimen with focus detection light (AF light) via the objective lens has an influence on the optical system (observation optical system) for generating an observation image in the microscope. In order to prevent this, the wavelength of the AF light is set to a wavelength (for example, near-infrared wavelength) deviating from the wavelength range of the observation light (visible light range).
JP 2004-70276 A

  However, it has been found that when fluorescence observation is performed using this focus detection apparatus, the focus detection accuracy may decrease depending on the type of fluorescent reagent that stains the specimen.

  Accordingly, an object of the present invention is to provide a fluorescence microscope apparatus capable of performing highly accurate focus detection regardless of the type of the fluorescence reagent, and a focus detection apparatus suitable for the fluorescence microscope apparatus.

  The fluorescence microscope apparatus of the present invention irradiates a fluorescent sample with excitation light through an objective lens, and detects the fluorescence generated in the fluorescent sample through the objective lens, and through the objective lens. A focus detection optical system that irradiates the fluorescent sample with focus detection light and detects the light reflected by the fluorescent sample through the objective lens, and the focus detection optical system irradiates and detects the light. And a first switching means for switching a focus detection wavelength, which is a wavelength of light, between at least two different wavelengths.

  A wavelength separation mirror is disposed at an intersection between the optical path of the observation optical system and the optical path of the focus detection optical system, and the first switching unit includes a wavelength separation mirror disposed at the intersection. Exchange may be made between at least two types of wavelength separation mirrors having different separation wavelengths.

  In the fluorescence microscope apparatus of the present invention, the combination of the excitation wavelength that is the wavelength of the excitation light that is irradiated by the observation optical system and the observation wavelength that is the wavelength of the fluorescence that is detected by the observation optical system are different from each other. You may further provide the 2nd switching means switched between at least 2 types of combinations.

  In addition, a wavelength separation mirror is disposed at an intersection between the excitation optical path and the observation optical path of the observation optical system, and the second switching unit converts the wavelength separation mirror disposed at the intersection at least with a different separation wavelength. You may exchange between two types of wavelength separation mirrors.

  The first switching means may be interlocked with the second switching means.

  In addition, one of the two types of wavelengths to which the focus detection wavelength is switched may be an infrared wavelength, and the other may be a visible light wavelength.

  The focus detection apparatus of the present invention irradiates a sample with focus detection light through an objective lens, and detects the light reflected by the sample through the objective lens, and the focus detection optical system, It is characterized by comprising switching means for switching a focus detection wavelength, which is a wavelength of light to be irradiated and detected by the focus detection optical system, between at least two different wavelengths.

  A wavelength separation mirror is disposed at the intersection between the optical path of the observation optical system used for observing the sample and the optical path of the focus detection optical system, and the switching means is disposed at the intersection. The wavelength separation mirror may be exchanged between at least two types of wavelength separation mirrors having different separation wavelengths.

  In addition, one of the two types of wavelengths to which the focus detection wavelength is switched may be an infrared wavelength, and the other may be a visible light wavelength.

  ADVANTAGE OF THE INVENTION According to this invention, the fluorescence microscope apparatus which can perform a highly accurate focus detection irrespective of the kind of fluorescence reagent used for the fluorescence sample, and a focus detection apparatus suitable for the fluorescence microscope apparatus are implement | achieved.

[Embodiment]
Hereinafter, embodiments of the fluorescence microscope apparatus will be described.

  FIG. 1 is a configuration diagram of a fluorescence microscope apparatus. As shown in FIG. 1, the fluorescence microscope apparatus includes an observation optical system 3, a focus detection optical system 2, a first objective lens 12, a stage 40, an input unit 43, a drive unit 108, and a drive unit 109. A drive unit 202, a CPU 41, and a memory 42. The first objective lens 12 may be exchangeable between a plurality of types of first objective lenses having different magnifications.

  A specimen 112 sandwiched between the slide glass 15 and the cover glass 14 is placed on the stage 40 as the object to be observed 0. The specimen 112 is a liquid containing a biological sample stained with any one of the fluorescent reagents A to D. Details of the fluorescent reagents A to D will be described later.

  The observation optical system 3 includes an observation illumination optical system 102, a wheel 101, and an observation imaging optical system 38.

  The wheel 101 has four wheel addresses 1 to 4, and different types of fluorescent filter blocks 104-1 to 104-4 prepared by the user are individually loaded in the wheel addresses 1 to 4. Details of these fluorescent filter blocks 104-1 to 104-4 will be described later.

  When the wheel 101 is driven by the drive unit 108, the effective wheel address is switched between the wheel addresses 1 to 4, and the fluorescent filter block inserted into the optical path of the observation optical system 3 is thereby switched to the fluorescent filter block 104-1. Switch between 104-4.

  Each of the fluorescent filter blocks 104-1 to 104-4 is provided with a barrier filter (BA filter) 106, an excitation filter (EX filter) 107, and a dichroic mirror 100 as shown in FIG.

  The focus detection optical system 2 includes a focus detection illumination optical system 5, a slider 201, and a focus detection imaging optical system 7.

  The slider 201 has two slider addresses 1 and 2, and the slider addresses 1 and 2 include AF filter blocks 105-1 and 105-2 of different types prepared by the manufacturer of the fluorescence microscope apparatus. Are installed individually. Details of these AF filter blocks 105-1 and 105-2 will be described later.

  When the slider 201 is driven by the drive unit 109, the effective slider address is switched between the slider addresses 1 and 2, and the AF filter block inserted into the optical path of the focus detection optical system 2 thereby becomes the AF filter block 105-. 1 and 105-2.

  Each of the AF filter blocks 105-1 and 105-2 is provided with an X filter 110, a Y filter 111, and a dichroic mirror 16 as shown in FIG.

  The input unit 43 illustrated in FIG. 1 is a mouse or a keyboard that is operated when a user inputs various instructions to the fluorescence microscope apparatus. One of the input instructions is an instruction to drive the wheel 101. When a driving instruction for the wheel 101 is input, a driving wheel address is also input.

  The input unit 43 is also used when the user inputs various data to the fluorescence microscope apparatus. One of the input data includes registration data of the wheel 101, that is, characteristic data of the fluorescent filter blocks 104-1 to 104-4 loaded in the wheel addresses 1 to 4. The CPU 41 shown in FIG. 1 controls each unit of the fluorescence microscope apparatus in accordance with instructions and data input from the input unit 43.

  FIG. 2 is a diagram showing in detail the optical system portion of the fluorescence microscope apparatus. In FIG. 2, the same elements as those shown in FIG. Here, the optical path when the wheel address 1 and the slider address 1 are effective (that is, the fluorescence filter block 104-1 and the AF filter block 105-1 are inserted in the optical path) will be described.

  As shown in FIG. 2, the observation optical system 3 includes a light source (for example, a mercury lamp, a white laser, etc.) 102a that emits light including at least the same wavelength component as the excitation wavelengths of the fluorescent reagents A to D, and a collector lens 102b. , 102c, a fluorescent filter block 104-1, a second objective lens 38a, a relay lens 38b, and an image sensor 38c having sensitivity in a wavelength region of at least 400 nm to 850 nm. Among these elements, the elements from the light source 102 a to the collector lens 102 c constitute the observation illumination optical system 102, and the elements from the second objective lens 38 a to the imaging element 38 c constitute the observation imaging optical system 38.

  On the other hand, the focus detection optical system 2 includes a light source (for example, a white LED) 5a that emits light including the same wavelength components as at least two types of AF light described later, a collector lens 5b, and a slit plate 5c. Collector lens 5d, pupil limiting mask 5e, half mirror 5f, AF filter block 105-1, second objective lens 7a, relay lens 7b, pupil limiting mask 7c, relay lens 7d, A cylindrical lens 7e and a line sensor 7d having sensitivity in at least two types of AF light wavelength ranges described later are disposed. An image sensor may be used instead of the line sensor. Among these elements, the elements from the light source 5a to the pupil restriction mask 5e constitute the focus detection illumination optical system 5, and the elements from the second objective lens 7a to the line sensor 7d constitute the focus detection imaging optical system 7.

  The light emitted from the light source 102a of the observation optical system 3 enters the EX filter 107 via the collector lenses 102b and 102c. A part of the wavelength component of the incident light passes through the EX filter 107 and enters the dichroic mirror 100 as excitation light. The excitation light is reflected by the dichroic mirror 100, passes through the Y filter 111, enters the dichroic mirror 16, and passes through the dichroic mirror 16. The excitation light enters the object to be observed 0 through the first objective lens 12 and excites the fluorescent substance contained in the specimen 112. The fluorescence generated in the sample 112 enters the dichroic mirror 16 through the first objective lens 12 and passes through the dichroic mirror 16. The fluorescence passes through the Y filter 111 and then enters the dichroic mirror 100 and passes through the dichroic mirror 100. The fluorescence passes through the BA filter 106, enters the image sensor 18c via the second objective lens 38a and the relay lens 38b, and forms a fluorescence image of the specimen 112 on the image sensor 18c.

  The output signal of the image sensor 18c is output to a monitor (not shown) via the CPU 41 in FIG. The user can observe the fluorescence image of the specimen 112 on the monitor.

  On the other hand, light emitted from the light source 5a of the focus detection optical system 2 enters the X filter 110 via the collector lens 5b, the slit plate 5c, the collector lens 5d, the pupil limiting mask 5e, and the half mirror 5f. A part of the wavelength component of the incident light passes through the X filter 110 and enters the dichroic mirror 16 as AF light. The AF light is reflected by the dichroic mirror 16, enters the object to be observed 0 through the first objective lens 12, and forms a slit image on the surface of the cover glass 14. The AF light reflected from the surface of the cover glass 14 enters the dichroic mirror 16 through the first objective lens 12 and is reflected by the dichroic mirror 16. The AF light passes through the X filter 110 and enters the line sensor 7d via the half mirror 5f, the second objective lens 7a, the relay lens 7b, the pupil limiting mask 7c, the relay lens 7d, and the cylindrical lens 7e. A slit image of the slit plate 5c is formed on the line sensor 7d.

  Here, the longitudinal direction of the line sensor 7d and the longitudinal direction of the slit image intersect, and the position of the slit image in the line sensor 7d is determined by the defocus amount of the first objective lens 12 (the focal plane of the objective lens and the sample surface). In the longitudinal direction of the line sensor 7d.

  The output signal of the line sensor 7d is converted into a defocus signal by a signal processing circuit (not shown) and then input to the drive unit 202 via the CPU 41 in FIG. The drive unit 202 adjusts the focus of the first objective lens 12 with respect to the object to be observed 0 by moving the stage 40 up and down according to the defocus signal.

  FIG. 3 is a diagram for explaining in detail the fluorescent filter blocks 104-1 to 104-4 loaded in the wheel addresses 1 to 4. As shown in FIG. The fluorescent filter block 104-1 is a fluorescent filter block used when the fluorescent reagent A is used, and the fluorescent filter block 104-2 is a fluorescent filter block used when the fluorescent reagent B is used. -3 is a fluorescent filter block used when the fluorescent reagent C is used, and a fluorescent filter block 104-4 is a fluorescent filter block used when the fluorescent reagent D is used.

4 is a diagram showing the characteristics of the fluorescent reagent A, FIG. 5 is a diagram showing the characteristics of the fluorescent reagent B, FIG. 6 is a diagram showing the characteristics of the fluorescent reagent C, and FIG. FIG. In each figure, show by symbol S _E is the absorption spectrum of the fluorescent reagent, indicate by reference numeral S _F is the emission spectrum of the fluorescent reagent.

As shown in FIG. 4 (and FIG. 3), the excitation wavelength (absorption spectrum S _E peak) of the fluorescent reagent A is 489 nm, and the fluorescence wavelength (emission spectrum S _F peak) is 508 nm.

As shown in FIG. 5 (and FIG. 3), the excitation wavelength (peak of absorption spectrum S _E ) of fluorescent reagent B is 514 nm, and the fluorescence wavelength (peak of emission spectrum S _F ) is 527 nm.

As shown in FIG. 6 (and FIG. 3), the excitation wavelength (peak of absorption spectrum S _E ) of fluorescent reagent C is 590 nm, and the fluorescence wavelength (peak of emission spectrum S _F ) is 615 nm.

As shown in FIG. 7 (and FIG. 3), the excitation wavelength (absorption spectrum S _E peak) of the fluorescent reagent D is 735 nm, and the fluorescence wavelength (emission spectrum S _F peak) is 779 nm.

As is clear from comparison of FIGS. 4, 5, 6, and 7, the absorption spectra S _E and emission spectra S _F of the fluorescent reagents A, B, and C are substantially within the visible light range (700 nm or less). On the other hand, the absorption spectrum S _E and the emission spectrum S _F of the fluorescent reagent D do not fall within the visible light region (700 nm or less), but extend to the near infrared region (above 700 nm). In the conventional focus detection apparatus, the focus detection accuracy is lowered when such a fluorescent reagent D is used.

  8 is a diagram showing the characteristics of the fluorescent filter block 104-1 loaded in the wheel address 1, and FIG. 9 is a diagram showing the characteristics of the fluorescent filter block 104-2 loaded in the wheel address 2. FIG. 11 is a diagram showing characteristics of the fluorescent filter block 104-3 loaded in the wheel address 3, and FIG. 11 is a diagram showing characteristics of the fluorescent filter block 104-4 loaded in the wheel address 4. In each figure, the symbol EX represents the spectral transmission characteristic of the EX filter 107, the symbol BA represents the spectral transmission characteristic of the BA filter 106, and the symbol DC represents the spectral transmission characteristic of the dichroic mirror 100. Yes (the wavelength range where the transmittance is zero in the spectral transmission characteristics of the dichroic mirror 100 is the reflection wavelength range).

Incidentally, the absorption spectrum S _E and an emission spectrum S _F of the fluorescent reagent A superimposed display in FIG. 8, superimposed to the absorption spectrum S _E and an emission spectrum S _F of fluorescent reagent B in FIG. 9, FIG. 10 fluorescent reagent C absorption spectrum S to superimpose _E and emission spectra S _F of superimposed displays an absorption spectrum S _E and an emission spectrum S _F of fluorescent reagent D in FIG. 11.

  As is clear from FIG. 8 (and FIG. 3), the EX filter 107 of the fluorescent filter block 104-1 selectively selects light in a wavelength range (460 nm to 500 nm) corresponding to the main part of the absorption wavelength range of the fluorescent reagent A. There is a transparent function. Further, the BA filter 106 of the fluorescence filter block 104-1 has a function of selectively transmitting light in a wavelength range (510 nm to 560 nm) corresponding to the main part of the emission wavelength range of the fluorescent reagent A. The separation wavelength of the dichroic mirror 100 of the fluorescent filter block 104-1 is set to a wavelength (505 nm) corresponding to the boundary between the main part of the absorption wavelength region of the fluorescent reagent A and the main part of the emission wavelength region of the fluorescent reagent A. Has been.

  Therefore, in the fluorescence filter block 104-1, the wavelength of the excitation light is set to a wavelength suitable for the fluorescent reagent A, and light (observation light) that can enter the observation imaging optical system 38 from the observation object 0 is observed. There is a function of setting the wavelength range to a wavelength range suitable for the fluorescent reagent A.

  9 (and FIG. 3), the EX filter 107 of the fluorescence filter block 104-2 selects light in a wavelength region (490 nm to 510 nm) corresponding to the main part of the absorption wavelength region of the fluorescence reagent B. Has a transparent function. The BA filter 106 of the fluorescence filter block 104-2 has a function of selectively transmitting light in a wavelength region (520 nm to 560 nm) corresponding to the main part of the emission wavelength region of the fluorescence reagent B. The separation wavelength of the dichroic mirror 100 of the fluorescent filter block 104-2 is set to a wavelength (515 nm) corresponding to the boundary between the main part of the absorption wavelength region of the fluorescent reagent B and the main part of the emission wavelength region of the fluorescent reagent B. ing.

  Therefore, in the fluorescence filter block 104-2, the wavelength of the excitation light is set to a wavelength suitable for the fluorescent reagent B, and light (observation light) that can enter the observation imaging optical system 38 from the observation object 0 is observed. There is a function of setting the wavelength range to a wavelength range suitable for the fluorescent reagent B.

  Further, as is clear from FIG. 10 (and FIG. 3), the EX filter 107 of the fluorescence filter block 104-3 selects light in a wavelength range (520 nm to 560 nm) corresponding to the main part of the absorption wavelength range of the fluorescence reagent C. Has a transparent function. Further, the BA filter 106 of the fluorescence filter block 104-3 has a function of selectively transmitting light in a wavelength region (600 nm to 660 nm) corresponding to the main part of the emission wavelength region of the fluorescent reagent C. The separation wavelength of the dichroic mirror 100 of the fluorescence filter block 104-3 is set to a wavelength (595 nm) corresponding to the boundary between the main part of the absorption wavelength region of the fluorescent reagent C and the main part of the emission wavelength region of the fluorescent reagent B. Has been.

  Therefore, in the fluorescent filter block 104-3, the wavelength of the excitation light is set to a wavelength suitable for the fluorescent reagent C, and light (observation light) that can enter the observation imaging optical system 38 from the observation object 0 is observed. There is a function of setting the wavelength range to a wavelength range suitable for the fluorescent reagent C.

  Further, as is clear from FIG. 11 (and FIG. 3), the EX filter 107 of the fluorescence filter block 104-4 selects light in a wavelength region (670 nm to 750 nm) corresponding to the main part of the absorption wavelength region of the fluorescence reagent D. Has a transparent function. Further, the BA filter 106 of the fluorescence filter block 104-4 has a function of selectively transmitting light in a wavelength range (770 nm to 850 nm) corresponding to the main part of the emission wavelength range of the fluorescent reagent D. The separation wavelength of the dichroic mirror 100 of the fluorescent filter block 104-4 is set to a wavelength (750 nm) corresponding to the boundary between the main part of the absorption wavelength region of the fluorescent reagent D and the main part of the emission wavelength region of the fluorescent reagent D. Has been.

  Therefore, in the fluorescence filter block 104-4, the wavelength of the excitation light is set to a wavelength suitable for the fluorescent reagent D, and light (observation light) that can enter the observation imaging optical system 38 from the observation object 0 is observed. There is a function of setting the wavelength range to a wavelength range suitable for the fluorescent reagent D.

  FIG. 12 is a diagram for explaining the AF filter blocks 105-1 and 105-2 loaded in the slider addresses 1 and 2 in detail.

  FIG. 13 is a diagram illustrating the characteristics of the AF filter block 105-1, and FIG. 14 is a diagram illustrating the characteristics of the AF filter block 105-2. In each figure, the symbol Y indicates the spectral transmission characteristic of the Y filter 111, the symbol X indicates the spectral transmission characteristic of the X filter 110, and the symbol DC indicates the spectral transmission characteristic of the dichroic mirror 16. There is a wavelength range in which the transmittance is zero in the spectral transmission characteristics of the dichroic mirror 16 is a reflection wavelength range.

  As is apparent from FIG. 13 (and FIG. 12), the X filter 110 of the AF filter block 105-1 has a function of selectively transmitting light in the near infrared region (730 nm to 770 nm). The dichroic mirror 16 of the AF filter block 105-1 has a function of reflecting light in the near infrared region (730 nm to 770 nm) and transmitting other light. The Y filter 111 of the AF filter block 105-1 has a function of selectively transmitting light in a wavelength region (700 nm or less) shorter than the near infrared region.

  Accordingly, the AF filter block 105-1 includes (1) the wavelength of the AF light is set in the near infrared region, and (2) light having a wavelength different from that of the AF light is emitted from the observation object 0 to the focus detection imaging optical system. 7, (3) prevent the AF light from entering the observation optical system 3, and (4) light having a wavelength different from that of the AF light between the object to be observed 0 and the observation optical system 3. There is a function that does not prevent people from coming and going. The AF filter block 105-1 having such characteristics should be used together with any of the fluorescent reagents A, B, and C.

  On the other hand, as is clear from FIG. 14 (and FIG. 12), the X filter 110 of the AF filter block 105-2 has a function of selectively transmitting light in the green light region (520 nm to 560 nm). Further, the dichroic mirror 16 of the AF filter block 105-2 has a function of reflecting light in the green light region (520 nm to 560 nm) and transmitting other light. The Y filter 111 of the AF filter block 105-2 has a function of selectively transmitting light outside the green light range (520 nm to 560 nm).

  Therefore, in the AF filter block 105-2, (1) the wavelength of the AF light is set in the green light range, and (2) light having a wavelength different from that of the AF light is transmitted from the observation object 0 to the focus detection imaging optical system 7. (3) prevent the AF light from entering the observation optical system 3, and (4) light having a wavelength different from that of the AF light between the object to be observed 0 and the observation optical system 3. There is a function that does not interfere with traffic. The AF filter block 105-2 having such characteristics is to be used together with the fluorescent reagent D.

  Here, if the AF filter block 105-1 is used when the fluorescent reagent D is used (that is, when the fluorescent filter block 104-4 is used), the observation light set by the fluorescent filter block 104-4 is used. , And the AF light wavelength range (730 nm to 770 nm) set by the AF filter block 105-1 overlap, the AF filter block 105-1 is a part of the observation light ( It is impossible to prevent the component having the same wavelength as the AF light) from entering the focus detection imaging optical system 7 together with the AF light. In this case, an unnecessary spot image (an unnecessary spot image that does not move in accordance with the defocus amount of the first objective lens 12) is formed on the line sensor 7d, so that the position of the slit image on the line sensor 7 is increased. It becomes impossible to detect with accuracy. This problem cannot be avoided with the conventional focus detection apparatus.

  However, since the fluorescence microscope apparatus of this embodiment can switch the AF filter block between the AF filter blocks 105-1 and 105-2, any one of the fluorescent reagents A, B, C, and D is used. If the switching is used such that the AF filter block 105-1 is sometimes used, and the AF filter block 105-2 is used when the fluorescent reagent D is used, the wavelength range of the observation light overlaps with the wavelength range of the AF light. Therefore, the focus detection accuracy does not deteriorate.

  Therefore, as long as the AF filter blocks 105-1 and 105-2 are properly switched, a focused fluorescent image is always generated regardless of the type of fluorescent reagent.

  FIG. 15 is a diagram showing the contents of the registration table stored in the memory 42. The registration table is created in advance by the CPU 41 based on registration data input by the user via the input unit 43.

  As illustrated in FIG. 15, the registration table stores information indicating the wavelength characteristics of the wheel addresses 1 to 4 and information indicating the appropriate slider addresses of the wheel addresses 1 to 4.

  The wavelength characteristic of the wheel address 1 is the separation wavelength of the dichroic mirror 100 of the fluorescent filter block 104-1 loaded in the wheel address 1, and in this embodiment, the separation wavelength is set to 505 nm. “505”.

  The wavelength characteristic of the wheel address 2 is the separation wavelength of the dichroic mirror 100 of the fluorescent filter block 104-2 loaded in the wheel address 2, and in this embodiment, the separation wavelength is set to 515 nm. “505”.

  The wavelength characteristic of the wheel address 3 is the separation wavelength of the dichroic mirror 100 of the fluorescent filter block 104-3 loaded in the wheel address 3, and in this embodiment, the separation wavelength is 595 nm, so the stored value of the memory 42 is “595”.

  The wavelength characteristic of the wheel address 4 is the separation wavelength of the dichroic mirror 100 of the fluorescent filter block 104-4 loaded in the wheel address 4, and in this embodiment, the separation wavelength is set to 750 nm. “750”.

  Further, the appropriate slider address of wheel address 1 is a slider address that should be valid when wheel address 1 is valid. In this embodiment, the fluorescent filter block 104-1 loaded in the wheel address 1 is used when the fluorescent reagent A is used, and the AF filter block used when the fluorescent reagent A is used is the AF filter block. 105-1 and the mounting destination is the slider address 1. Therefore, the appropriate slider address of wheel address 1 is “1”.

  Further, the appropriate slider address of the wheel address 2 is a slider address that should be valid when the wheel address 2 is valid. In this embodiment, the fluorescent filter block 104-2 loaded in the wheel address 2 is used when the fluorescent reagent B is used, and the AF filter block used when the fluorescent reagent B is used is the AF filter block. 105-1 and the mounting destination is the slider address 1. Therefore, the appropriate slider address of wheel address 2 is also “1”.

  The appropriate slider address of the wheel address 3 is a slider address that should be valid when the wheel address 3 is valid. In the present embodiment, the fluorescent filter block 104-3 loaded in the wheel address 3 is used when the fluorescent reagent C is used, and the AF filter block used when the fluorescent reagent C is used is the AF filter block. 105-1 and the mounting destination is the slider address 1. Therefore, the appropriate slider address of the wheel address 3 is also “1”.

  The appropriate slider address of the wheel address 4 is a slider address that should be valid when the wheel address 4 is valid. In this embodiment, the fluorescent filter block 104-4 loaded in the wheel address 4 is used when the fluorescent reagent D is used, and the AF filter block used when the fluorescent reagent D is used is the AF filter block. 105-2 and the mounting destination is the slider address 2. Therefore, the appropriate slider address of the wheel address 4 is “2”.

  FIG. 16 is an operation flowchart of the CPU 41 regarding the driving of the wheel 101. Hereinafter, each step will be described in order.

  Step S11: The CPU 41 determines whether or not a driving instruction for the wheel 101 and a driving destination wheel address have been input from the user. The user inputs “1” as the driving destination wheel address when using the fluorescent reagent A, inputs “2” as the driving destination wheel address when using the fluorescent reagent B, and the driving destination when using the fluorescent reagent C. “3” is input as the wheel address, and “4” is input as the drive destination wheel address when the fluorescent reagent D is used. The CPU 41 proceeds to step S12 when the drive instruction and the drive wheel address are input, and waits when it is not input.

  Step S12: The CPU 41 checks the currently valid wheel address (drive source wheel address) via the drive unit 108, and on the registration table stored in the memory 42, the appropriate slider associated with the drive source wheel address. Check the address (A).

  Step S13: The CPU 41 checks the appropriate slider address (B) associated with the driving wheel address input by the user on the registration table stored in the memory 42.

  Step S14: The CPU 41 determines whether or not the appropriate slider address (A) and the appropriate slider address (B) are the same. If they are the same, the process proceeds to step S16, and if they are different, the process proceeds to step S15. .

  Step S15: The CPU 41 drives the slider 201 by giving an instruction to the drive unit 109, and after making the appropriate slider address (B) valid, the process proceeds to step S16.

  Step S16: The CPU 41 drives the wheel 101 by giving an instruction to the drive unit 108, validates the drive destination wheel address, and ends the flow.

  According to the above flow, the switching of the fluorescence filter blocks 104-1 to 104-4 and the switching of the AF filter blocks 105-1 and 105-2 are linked. Specifically, when any one of the fluorescent filter blocks 104-1 to 104-3 is inserted into the optical path, the AF filter block 105-1 is automatically inserted into the optical path, and the fluorescent filter block 104-4 is inserted into the optical path. When this is done, the AF filter block 105-2 is automatically inserted into the optical path.

  Therefore, the user of the fluorescence microscope apparatus can always observe a focused fluorescent image regardless of the type of the fluorescent reagent, as long as the switching of the fluorescent filter blocks 104-104 to 104-4 is appropriately performed.

  FIG. 17 is an operation flowchart of the CPU 41 relating to the creation of the registration table. Hereinafter, each step will be described in order.

  Step S21: The CPU 41 sets the address number i to an initial value (1).

Step S22: The CPU 41 allows the user to input the numerical value of the separation wavelength l _i of the dichroic mirror 100 of the fluorescent filter block 104-i loaded at the wheel address i. The CPU 41 may input the type of the fluorescent filter block instead of inputting a numerical value, and estimate the separation wavelength from the type.

Step S23: CPU 41 estimates the maximum wavelength L _i of the observation light which is set when the wheel address i is valid, for example, by the equation of L = l _i + 100nm.

Step S24: The CPU 41 compares the estimated longest wavelength L _i with the shortest wavelength L _AF (730 nm in the present embodiment) of the AF light that is set when the slider address 1 is validated. If is smaller, the process proceeds to step S25, and if the latter is smaller, the process proceeds to step S26.

  Step S25: The CPU 41 sets the appropriate slider address of the wheel address i to “1”, and proceeds to step S27.

  Step S26: The CPU 41 sets the appropriate slider address of the wheel address i to “2”, and proceeds to step S27.

  Step S27: The CPU 41 determines whether or not the number of addresses i has reached the maximum value “4”. If not, the process proceeds to step S28, and if it has reached, the flow ends.

  Step S28: The CPU 41 increments the address number i by 1 and returns to step S22.

In the above flow, when the address number i is “1”, “505 nm” is input as the numerical value of the separation wavelength l _i , so the longest wavelength L _i of the observation light is estimated to be “605 nm”. In this case, since L _i < _LAF is satisfied, the appropriate slider address is “1”.

In the above flow, when the address number i is “2”, “515 nm” is input as the numerical value of the separation wavelength l _i , so the longest wavelength L _i of the observation light is estimated to be “615 nm”. In this case as well, L _i < _LAF is satisfied, so the appropriate slider address is “1”.

Further, in the above flow, when the number of addresses i is “3”, “595 nm” is input as the numerical value of the separation wavelength l _i , so the longest wavelength L _i of the observation light is estimated to be “695 nm”. In this case as well, L _i < _LAF is satisfied, so the appropriate slider address is “1”.

In the above flow, when the address number i is “4”, “750 nm” is input as the numerical value of the separation wavelength l _i , so the longest wavelength L _i of the observation light is estimated to be “850 nm”. In this case, since L _i < _LAF does not hold, the appropriate slider address is not “1” but “2”. That is, according to the flow of FIG. 17, the registration table shown in FIG. 15 is automatically created.

  Therefore, the user of the fluorescence microscope apparatus can always observe a focused fluorescent image regardless of the type of the fluorescent reagent as long as the registration data regarding the wheel 101 is appropriately input.

[Supplement of embodiment]
In the embodiment described above, the settable AF light wavelength is a combination of near-infrared light and green light, but a combination of near-infrared light and other visible light, for example, near-infrared light and A combination with purple light, a combination of near infrared light and blue light, a combination of near infrared light and orange light, or the like may be used. However, it is desirable that the visible light combined with the near-infrared light is light having a wavelength somewhat away from the near-infrared light.

  In the above-described embodiment, one of the wavelengths of AF light that can be set is near-infrared light, but both wavelengths of AF light that can be set may be visible light. However, also in this case, it is desirable that the wavelengths of the two are separated to some extent.

  In the above-described embodiment, the number of AF light wavelengths is switched to 2, but may be 3 or more.

  In the embodiment described above, it is desirable that the calculation content by the signal processing circuit is also switched when the wavelength of the AF light is switched. This is because when the wavelength of the AF light changes, the relationship between the defocus amount and the amount of movement of the slit image changes slightly.

  In the focus detection optical system according to the above-described embodiment, the pattern projected onto the object to be observed 0 is the slit, but may be replaced with a pattern other than the slit. However, even in such a case, it is desirable to select a pattern whose position movement amount is easy to detect, such as a cross pattern.

  In addition, although the slit projection method is applied to the focus detection optical system of the above-described embodiment, other methods that perform focus detection using AF light having a wavelength different from the observation light may be applied.

  In the above-described embodiment, the wavelength of the AF light is switched by a combination of one light source 5a and a plurality of AF filter blocks, but may be performed by a plurality of single wavelength light sources having different wavelengths.

  However, even in this case, it is possible to prevent light having a wavelength different from that of the AF light from traveling from the object to be observed 0 to the focus detection imaging optical system 7 and to allow the AF light to enter the observation optical system 3. In order to prevent this, it is desirable to switch between a plurality of AF filter blocks.

  In the above-described embodiment, the switching of the fluorescence filter block and the switching of the AF filter block are each electrically performed, but one or both of them may be manually performed. Even when the switching of the fluorescent filter block is performed manually, the switching of the AF filter block may be interlocked with the switching. Further, as the interlock, either an electrical interlock or a mechanical interlock may be adopted.

  In the above-described embodiment, the number of AF filter blocks is smaller than the number of fluorescent filter blocks. However, an AF filter block may be prepared for each fluorescent filter. In that case, each fluorescent filter is fixed in advance to an appropriate AF filter block, and the drive unit 109 is omitted, and the drive unit 108 may drive both the fluorescent filter block and the AF filter block together. . In this case, the configuration of the fluorescence microscope can be simplified, and the processing of the CPU 41 (FIG. 16) related to the interlock can be omitted.

It is a block diagram of a fluorescence microscope apparatus. It is a figure which shows the optical system part of a fluorescence microscope apparatus in detail. It is a figure explaining in detail four kinds of fluorescent filter blocks 104-1 to 104-4 loaded in wheel addresses 1 to 4. It is a figure which shows the characteristic of the fluorescence reagent A. It is a figure which shows the characteristic of the fluorescence reagent B. FIG. 4 is a diagram showing characteristics of a fluorescent reagent C. FIG. 5 is a diagram showing characteristics of a fluorescent reagent D It is a figure which shows the characteristic of the fluorescence filter block 104-1. It is a figure which shows the characteristic of the fluorescence filter block 104-2. It is a figure which shows the characteristic of the fluorescence filter block 104-3. It is a figure which shows the characteristic of the fluorescence filter block 104-4. It is a figure explaining AF filter block 105-1 and 105-2 loaded in slider address 1 and 2 in detail. It is a figure which shows the characteristic of AF filter block 105-1. It is a figure which shows the characteristic of AF filter block 105-2. It is a figure which shows the content of the registration table which the memory 42 has stored. 3 is an operation flowchart of a CPU 41 related to driving of a wheel 101. It is an operation | movement flowchart of CPU41 regarding preparation of a registration table.

Explanation of symbols

DESCRIPTION OF SYMBOLS 2 ... Optical system for focus detection, 3 ... Optical system for observation, 5 ... Illumination optical system for focus detection, 7 ... Imaging optical system for focus detection, 102 ... Illumination optical system for observation, 38 ... Imaging optical system for observation , 12 ... first objective lens, 40 ... stage, 0 ... observed object, 15 ... slide glass, 14 ... cover glass, 112 ... specimen, 101 ... wheel, 104 ... fluorescence filter block, 201 ... slider, 105 ... AF filter Block 106, barrier filter (BA filter), 107 excitation filter (EX filter), 100 dichroic mirror, 110 X filter, 111 Y filter, 16 dichroic mirror

Claims (9)

An observation optical system for irradiating the fluorescent sample with excitation light through the objective lens and detecting the fluorescence generated in the fluorescent sample through the objective lens;
A focus detection optical system that irradiates the fluorescent sample with light for focus detection through the objective lens and detects the light reflected by the fluorescent sample through the objective lens;
A first switching means for switching a focus detection wavelength, which is a wavelength of light to be irradiated and detected by the focus detection optical system, between at least two different wavelengths;
A fluorescence microscope apparatus comprising: The fluorescence microscope apparatus according to claim 1,
A wavelength separation mirror is disposed at an intersection between the optical path of the observation optical system and the optical path of the focus detection optical system,
The first switching means includes
The fluorescence microscope apparatus characterized by exchanging the wavelength separation mirrors arranged at the intersections between at least two types of wavelength separation mirrors having different separation wavelengths. In the fluorescence microscope apparatus according to claim 1 or 2,
A combination of an excitation wavelength, which is a wavelength of excitation light emitted by the observation optical system, and an observation wavelength, which is a fluorescence wavelength detected by the observation optical system, is switched between at least two different combinations. A fluorescence microscope apparatus further comprising two switching means. The fluorescence microscope apparatus according to claim 3,
A wavelength separation mirror is disposed at the intersection of the excitation optical path and the observation optical path of the observation optical system,
The second switching means includes
The fluorescence microscope apparatus characterized by exchanging the wavelength separation mirrors arranged at the intersections between at least two types of wavelength separation mirrors having different separation wavelengths. In the fluorescence microscope apparatus according to claim 3 or 4,
The first switching means includes
A fluorescence microscope apparatus that is linked to the second switching means. In the fluorescence microscope apparatus according to any one of claims 1 to 5,
One of the two types of wavelengths to which the focus detection wavelength is switched is an infrared wavelength, and the other is a visible light wavelength. A focus detection optical system that irradiates the sample with focus detection light through the objective lens and detects the light reflected by the sample through the objective lens;
A focus detection apparatus comprising: switching means for switching a focus detection wavelength, which is a wavelength of light to be irradiated and detected by the focus detection optical system, between at least two different wavelengths. The focus detection apparatus according to claim 7,
A wavelength separation mirror is disposed at the intersection of the optical path of the observation optical system used for observation of the sample and the optical path of the focus detection optical system,
The switching means is
The focus detection apparatus, wherein the wavelength separation mirrors arranged at the intersection are exchanged between at least two types of wavelength separation mirrors having different separation wavelengths. In the focus detection apparatus according to claim 7 or 8,
One of the two types of wavelengths to which the focus detection wavelength is switched is an infrared wavelength, and the other is a visible light wavelength.

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