JP4451110B2 - Microscope system - Google Patents

Description

  The present invention relates to a microscope system used for observing a specimen labeled with a fluorescent dye.

  Conventionally, there is a microscope system in which a fluorescent dye is irradiated with light having a predetermined wavelength to excite the fluorescent dye, and the fluorescence emitted by this excitation is observed.

  In this case, there are a plurality of fluorescent dyes for fluorescently labeling the specimen, and the excitation light wavelength and the fluorescence wavelength are different depending on the fluorescent dye. For example, fluorescent dyes such as Fitc that is excited at a laser wavelength of 488 nm and emits fluorescence at around 530 nm and Tritc that is excited at a laser wavelength of 543 nm and emits fluorescence at around 580 nm are known, and these are fluorescently labeled. It is properly used depending on the cell site. In some cases, multiple stained specimens in which a single specimen is labeled with a plurality of fluorescent dyes are observed simultaneously or by switching.

  On the other hand, when performing fluorescence observation using such multiple fluorescent dyes, in order to efficiently separate excitation light and fluorescence, multiple types of excitation dichroic mirrors with spectral characteristics suitable for each fluorescent dye and observation method are used. It is necessary to prepare. In this case, these excitation dichroic mirrors are used by switching optimal ones on the optical path, and those described in Patent Document 1 and Patent Document 2 are known as switching mechanisms therefor. These switching mechanisms disclosed in Patent Documents 1 and 2 are composed of a rotary turret, and a plurality of excitation dichroic mirrors arranged at each position of the turret can be switched on the optical path. Further, the excitation dichroic mirror and the absorption filter are configured as one cube unit, and this cube unit can be removed from the turret and replaced.

  Recently, in such observation, it has been considered to use illumination utilizing total reflection (hereinafter referred to as evanescent illumination) as a method for exciting a fluorescent dye. This is because the illumination range is extremely shallow with respect to the depth direction of the sample, so that information near the interface between the sample and the cover glass can be obtained with high sensitivity without being affected by the background.

  Patent Document 3 describes evanescent illumination. The evanescent illumination is performed by condensing laser light at a position shifted from the optical axis center of the rear focal position (pupil position) of the objective lens and illuminating the sample with parallel light from an oblique direction. In order to generate evanescent illumination, it is necessary to illuminate at an illumination angle larger than the angle at which total reflection occurs between the cover glass and the sample (greater than the critical angle), and the conditions are as follows.

n ₁ Xsinθ ≧ n ₂
However, n ₁ : refractive index of the immersion medium of the cover glass and the objective lens, θ: illumination angle, n ₂ : refractive index of the sample The relationship between the illumination angle θ and the illumination NA of the objective lens is as follows.

NA = n ₁ Xsinθ
Therefore, the conditions for causing the total reflection illumination are expressed as follows by the illumination NA of the objective lens.

NA ≧ n ₂
In addition, when the illumination angle is larger than the NA value (NAob) of the objective lens, light rays are scattered at the objective lens frame and at the end of the objective lens, so it is necessary to illuminate with an NA smaller than NAob.

  From the above, in order to perform evanescent illumination accurately, it is necessary to irradiate the sample with illumination NA that satisfies the following conditions.

NAob ≧ NA ≧ n ₂
JP-A-56-19605 Japanese Utility Model Publication No. 61-36966 JP 2001-272606 A

  However, when evanescent illumination is applied as excitation light to a method of performing observation by switching an excitation dichroic mirror suitable for a specimen labeled with a different fluorescent dye on the optical path, the following inconvenience occurs.

  The exchanging or exchanging place of the excitation dichroic mirror is shared with the part used in the optical path of the epi-illumination of a normal microscope using a mercury lamp as a light source. This is because the laser light used for evanescent illumination is introduced from the middle of the epi-illumination tube that constitutes the optical path of the epi-illumination of ordinary microscopes, and the optical path of the laser light and the illumination optical path of the ordinary microscope are shared. This is because the mirror switching mechanism can also be used.

  However, the switching mechanism of the excitation dichroic mirror has various errors such as fluctuations in the rotation center axis of the turret, variation in click position accuracy that determines the rotation direction, angle difference of the surface where the cube unit excitation dichroic mirror is mounted, folding position difference, etc. There is a difference in the reflection angle and the folding position due to switching or exchange of the excitation dichroic mirror.

  These deviations in reflection angle and folding position do not pose any problem in the incident-light fluorescent illumination of a normal microscope. However, when using evanescent illumination, the conditional expression for causing the above-described total reflection illumination is not satisfied depending on the deviation in illumination angle. In some cases, there is a problem that accurate evanescent illumination is not possible.

  As described above, when the excitation dichroic mirror of a normal microscope is also used as evanescent illumination, the displacement at the time of switching and replacement of the excitation dichroic mirror becomes a problem in the evanescent illumination. Therefore, as a countermeasure, it is conceivable to change the excitation dichroic mirror switching mechanism to one having higher accuracy than that used in a normal microscope. However, providing such a special excitation dichroic mirror switching mechanism is very expensive and impractical.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a microscope system capable of always performing accurate evanescent illumination.

  According to a first aspect of the present invention, there is provided a light source for generating laser light having a plurality of wavelengths and a light source for generating evanescent illumination by condensing the laser light from the light source on a plane perpendicular to the optical axis including the pupil of the objective lens. An optical optical system, an observation optical system for observing fluorescence generated from the sample by the evanescent illumination, and a switchable or exchangeable for each wavelength of the laser light of the light source, reflect the laser light, A plurality of types of wavelength-selective optical elements having a property of transmitting fluorescence; and a correction unit that corrects a deviation of a condensing position of the laser light reflected by each of the plurality of wavelength-selective optical elements at the objective lens. It is characterized by having.

  According to a second aspect of the present invention, in the first aspect of the invention, the correction unit includes a storage unit that stores a correction value according to an error related to the wavelength selection optical element, By exchanging, the deviation of the condensing position of the laser light from the light source at the objective lens is corrected based on the correction value stored in the storage means.

  According to a third aspect of the present invention, the optical system according to the second aspect of the present invention has a fiber for deriving laser light from the light source, and the condensing optical system has a laser light emitting end of the fiber at the objective lens. Projecting onto a plane perpendicular to the optical axis including the pupil, the correction means includes moving means for horizontally moving the laser light emitting end of the fiber.

  According to a fourth aspect of the present invention, in the second aspect of the invention, there is provided a deflection mirror that deflects the laser light from the light source, and the deflection mirror is deflected to two-dimensionally scan the sample with a light spot. A confocal observation function, wherein the condensing optical system is perpendicular to an optical axis including a first state in which the laser beam deflected by the deflection mirror is spot-illuminated on the sample and the pupil of the objective lens The second state of condensing light onto a smooth surface is switchable, and the correction means is performed by controlling the deflection angle of the deflection mirror in the second state.

  A fifth aspect of the present invention is the cube turret according to any one of the first to fourth aspects, wherein the wavelength selection optical element is formed of an excitation dichroic mirror mounted on a cube unit. Thus, it is possible to switch the excitation dichroic mirror.

  According to a sixth aspect of the present invention, in the fifth aspect of the present invention, the correction means determines a correction value of the storage means from the relationship between the cube unit and the position of the cube turret to which the cube unit is mounted. It is characterized by that.

  According to the present invention, when performing fluorescence observation using a plurality of fluorescent dyes, in order to efficiently separate excitation light and fluorescence, wavelength selective optics that is arranged and used on the optical path according to each fluorescent dye. Since the focus position shift on the pupil plane of the objective lens caused by switching or exchange of elements can be corrected for each wavelength selection optical element, the illumination NA of evanescent illumination can always be kept constant and stable. It is possible to realize total reflection fluorescence observation that enables evanescent illumination.

  Further, according to the present invention, the deviation of the condensing position of the laser light at the objective lens is corrected based on the correction value of the storage means that stores the correction value corresponding to the error relating to the wavelength selection optical element in advance. Highly accurate correction can be performed, and accurate evanescent illumination can be performed.

  Furthermore, according to the present invention, since the deviation of the focusing position of the objective lens can be corrected by using a deflection mirror used for confocal observation with a scanning laser microscope, the system can be reduced in size and priced. Even cheaper.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 shows a schematic configuration of a microscope system to which the present invention is applied.

  In the figure, reference numeral 1 denotes a laser light source unit as a light source. The laser light source unit 1 includes an argon (Ar) laser 2 that oscillates a 488 nm laser beam used for evanescent illumination and a green helium neon laser that oscillates a 543 nm laser beam. 3.

  A reflection mirror 4 is disposed on the optical path of the laser light from the green helium neon laser 3. Further, a dichroic mirror 5 is disposed on the intersection of the laser beam reflected by the reflection mirror 4 on the optical path of the laser beam from the argon laser 2. The dichroic mirror 5 combines these two laser light paths, and transmits the laser light from the argon laser 2 and reflects the laser light reflected by the reflection mirror 4. That is, the dichroic mirror 5 here has a characteristic of reflecting the laser beam of 543 nm and transmitting the laser beam of 488 nm.

  On the optical path of the laser beam synthesized by the dichroic mirror 5, an acoustooptic element (AOTF) 6 for wavelength selection is arranged. The acoustooptic device 6 here selects wavelengths of 488 nm and 543 nm.

  An incident end of a fiber 7 is disposed at the exit end of the acoustooptic device 6, and the laser light for evanescent illumination is guided through this fiber 7 to an evanescent light projecting tube 8 as an epi-illumination light projecting tube. .

  The evanescent light projecting tube 8 includes a linear light projecting tube main body 801 and a light guide tube 802 protruding in a direction orthogonal to the light projecting tube main body 801. One end of the light projecting tube main body 801 is an inverted microscope. It is fixed to the incident light projection tube mounting portion of the main body 9 with screws (not shown).

  The evanescent light projecting tube 8 is provided with an epi-illumination light source 12 at the other end of the light projecting tube main body 801. As the epi-illumination light source 12, for example, a mercury lamp is used for epi-illumination of an ordinary microscope.

  A laser introducing portion 8 a is provided at the tip of the light guide tube 802 of the evanescent projector tube 8. The exit end of the fiber 7 is connected to the laser introduction portion 8a. The laser introduction part 8a is mounted on an electric stage 8b as a moving means. The electric stage 8b is movable by an electric mechanism (not shown), and the emission end face of the fiber 7 is horizontally moved in the direction of the arrow 8d in the figure, that is, along the plane orthogonal to the optical axis of the laser beam emitted from the end of the fiber 7. It can move in the direction.

  On the optical path of the laser beam emitted from the end of the fiber 7, a reflection mirror 11 is disposed via a lens 10. The reflection mirror 11 is disposed at the intersection of the optical path of the illumination light of the epi-illumination light source 12 and the optical path of the laser light from the end of the fiber 7, and can be inserted into and removed from the optical path by a switching mechanism (not shown). That is, the reflection mirror 11 switches the optical path between the evanescent illumination from the fiber 7 and the normal epifluorescence illumination from the epi-illumination light source 12 by insertion / removal from the optical path. In the illustrated example, it is assumed that the reflecting mirror 11 is switched to a state where it is inserted into the optical path, that is, a state where evanescent illumination is performed.

  A condensing lens 13 constituting a condensing optical system is disposed in the reflection optical path of the reflection mirror 11. The condenser lens 13 projects the output end face of the fiber 7 to a position 14b offset from the center of the pupil position 14a on the plane perpendicular to the optical axis including the pupil of the objective lens 14 described later.

  A cube turret 15 is disposed in the optical path of the laser light emitted from the condenser lens 13. The cube turret 15 can hold four types of cube units, and can be rotated and switched by a motor 18 via a sliding mechanism such as a bearing (not shown).

  In the illustrated example, only two types of cube units 16 and 17 are mounted, and the cube unit 16 is positioned on the optical path of the laser light from the condenser lens 13.

  In this case, the cube unit 16 transmits, as a wavelength selection optical element, an excitation dichroic mirror 16a that reflects 488 nm and transmits 500 nm or more, and a fluorescence wavelength generated by cutting 488 nm and irradiating 488 nm. A barrier filter 16b is provided. In addition, the cube unit 17 is a wavelength selective optical element that reflects the light of 543 nm and transmits the light of 560 nm or more, and a barrier that transmits the fluorescence wavelength generated by cutting 543 nm and irradiating 543 nm. A filter 17b is provided. These cube units 16 and 17 can be removed from the cube turret 15 and replaced with other cube units.

  The objective lens 14 is disposed in the reflected light path of the excitation dichroic mirror 16a of the cube unit 16 (positioned on the optical path of the laser light from the condenser lens 13).

  A fluorescently labeled sample 21 is disposed at the focal position of the objective lens 14. The sample 21 is fixed to a cover glass 20 disposed on the sample stage 19 of the inverted microscope body 9. In this case, the cover glass 20 and the objective lens 14 are filled with immersion oil for securing a high NA and causing total reflection by evanescent illumination. The refractive index of the cover glass 20 and oil here is approximately 1.52.

  In the transmission optical path 9a of the excitation dichroic mirror 16a of the cube unit 16 (positioned on the optical path of the laser light from the condenser lens 13), an imaging lens 22 and a reflection mirror 23 that constitute the observation optical system are arranged. ing.

  The reflection mirror 23 can be inserted and removed from the optical path by a switching mechanism (not shown). In this case, the insertion / removal of the reflection mirror 23 onto / from the optical path may be performed alone or in conjunction with the insertion / removal of the reflection mirror 11 into / from the optical path. In the illustrated example, a state where the reflection mirror 23 is inserted in the optical path is shown.

  In a state where the reflection mirror 23 is inserted on the optical path, a CCD camera 24 as an imaging unit is disposed in the reflection optical path 9b of the reflection mirror 23. The fluorescence from the sample 21 that has passed through the imaging lens 22 is guided to the CCD camera 24 and imaged on the imaging surface 24a. Further, the reflection mirror 25 and the visual observation unit 26 are arranged in the visual observation optical path 9c in a state where the reflection mirror 23 is off the optical path. The fluorescence from the sample 21 that has passed through the imaging lens 22 is guided to the visual observation unit 26 and visually observed.

  On the other hand, a controller 27 is connected to the acoustooptic device 6, the electric stage 8 b that drives the emission end face of the fiber 7, and the motor 18 that rotationally drives the cube turret 15. The control unit 27 outputs control signals to the acoustooptic device 6, the electric stage 8 b and the motor 18. The control unit 27 is provided with a storage unit 27a as a storage unit.

  The storage unit 27a is used for the angle error and the folding position error of the mounting surfaces of the excitation dichroic mirrors of the cube units 16 and 17 mounted on the cube turret 15 and the cube units (not shown) prepared for replacement. Data for correcting the deviation of the projection position of the exit end face of the fiber 7 to the pupil position 14a of the objective lens 14 is stored. Here, the cube units 16 and 17 are placed in advance at the rotational position of the cube turret 15. First, each cube unit prepared for replacement is mounted, and the amount of deviation of the projection position of the exit end face of the fiber 7 at this time onto the pupil position 14a of the objective lens 14 is measured and corrected from this measurement result. The value is obtained and stored.

  When the cube turret 15 is actually mounted on the cube turret 15, the correction value is read from the storage unit 27a from the relationship with the rotational position of the cube turret 15 to which the cube units 16 and 17 are mounted. By using the value to control the amount of movement of the electric stage 8b, the position 14b in which the exit end of the fiber 7 is offset from the center of the pupil position 14a of the objective lens 14 (the illumination NA is suitable for performing evanescent illumination) Position).

  Next, the operation of the first embodiment configured as described above will be described.

  Now, it is assumed that the sample 21 is labeled with a fluorescent dye Fitc that is excited at 488 nm and emits fluorescence at about 520 to 580 nm.

  Then, the control unit 27 instructs the motor 18 to rotationally drive the cube turret 15 so that the cube unit 16 is positioned on the optical path. Further, the acoustooptic device 6 is instructed to select laser light having a wavelength of 488 nm oscillated from the argon laser 2 of the laser light source unit 1.

  Further, the control unit 27 reads a correction value from the storage unit 27a based on the relationship between the rotational position of the cube unit 16 and the cube turret 15 to which the cube unit 16 is mounted, and uses this correction value to move the electric stage 8b horizontally. The amount is controlled so that the output end of the fiber 7 is accurately projected to a position 14b (position where the illumination NA is suitable for performing evanescent illumination) offset from the center of the pupil position 14a of the objective lens 14. .

  In this state, the laser wavelength 488 nm oscillated from the argon laser 2 passes through the dichroic mirror 5 and the acoustooptic device 6 and is guided to the incident end of the fiber 7.

  The laser light emitted from the fiber 7 is guided to the cube unit 16 through the lens 10, the reflection mirror 11, and the condenser lens 13 of the evanescent light projection tube 8.

  Since the excitation dichroic mirror 16a mounted on the cube unit 16 reflects 488 nm, the laser light is reflected upward and condensed at a position 14b offset from the center of the pupil position 14a of the objective lens 14. Then, the objective lens 14 evanescently illuminates the sample 21 fluorescently labeled with Fitc.

  In this case, the exit end face of the fiber 7 is electrically driven based on the correction value stored in the storage unit 27a in order to correct errors caused by the angle error of the mounting surface of the excitation dichroic mirror 16a of the cube unit 16 and the folding position error. Since it is positioned by the stage 8b, accurate evanescent illumination is possible.

  Fitc fluorescence (520 to 580 nm) emitted from the sample 21 by such evanescent illumination passes through the objective lens 14, passes through the excitation dichroic mirror 16 a and the barrier filter 16 b in the cube unit 16, and enters the imaging lens 22. Incident. Then, the light is reflected by the reflection mirror 23 and formed on the image pickup surface 24a of the CCD camera 24, and a fluorescence observation image by evanescent illumination is picked up.

  When the reflection mirror 23 is removed from the optical path, the fluorescence transmitted through the imaging lens 22 is reflected by the reflection mirror 25 and visually observed by the visual observation unit 26.

  Next, it is assumed that the sample 21 is exchanged with one that is excited with 543 nm and labeled with a fluorescent dye Tritc that emits fluorescence at about 560 to 620 nm.

  Then, the control unit 27 instructs the motor 18 to rotationally drive the cube turret 15 so that the cube unit 17 is positioned on the optical path. Further, the acoustooptic device 6 is instructed to select the wavelength 543 nm oscillated from the green helium neon laser 3 of the laser light source unit 1.

  Further, the control unit 27 reads a correction value from the storage unit 27a based on the relationship between the rotation position of the cube unit 17 and the cube turret 15 to which the cube unit 17 is mounted, and uses this correction value to move the electric stage 8b horizontally. The amount is controlled so that the output end of the fiber 7 is accurately projected to a position 14b (position where the illumination NA is suitable for performing evanescent illumination) offset from the center of the pupil position 14a of the objective lens 14. .

  In this state, the laser wavelength 543 nm oscillated from the green helium neon laser 3 passes through the reflection mirror 4, the dichroic mirror 5, and the acoustooptic device 6 and is guided to the incident end of the fiber 7.

  The laser light emitted from the fiber 7 is guided to the cube unit 17 through the lens 10, the reflection mirror 11, and the condenser lens 13.

  Since the excitation dichroic mirror 17a mounted on the cube unit 17 reflects 543 nm, the laser light is reflected upward and is condensed at a position 14b offset from the center of the pupil position 14a of the objective lens 14. Then, the objective lens 14 illuminates the sample 21 fluorescently labeled with Tric.

  Also in this case, the emission end face of the fiber 7 is based on the correction value stored in the storage unit 27a in order to correct errors caused by the angle error of the mounting surface of the excitation dichroic mirror 17a of the cube unit 17 and the folding position error. Since it is positioned by the electric stage 8b, accurate evanescent illumination becomes possible.

  Tric fluorescence (560 to 620 nm) emitted from the sample 21 by such evanescent illumination passes through the objective lens 14, passes through the excitation dichroic mirror 17 a and the barrier filter 17 b in the cube unit 17, and enters the imaging lens 22. Incident. Then, the light is reflected by the reflection mirror 23 and formed on the image pickup surface 24a of the CCD camera 24, and a fluorescence observation image by evanescent illumination is picked up.

  When the reflection mirror 23 is removed from the optical path, the fluorescence transmitted through the imaging lens 22 is reflected by the reflection mirror 25 and visually observed by the visual observation unit 26.

  FIG. 2 is an enlarged view of the main part of FIG. 1 and explains in more detail the state of evanescent illumination that changes depending on the angle difference when the excitation dichroic mirror is switched.

In this case, for example, the NA value (NAob) of the objective lens 14 is 1.46, the focal length fob is 3 mm, the refractive index n ₂ of the sample 21 is 1.38, and the evanescent first set by the dichroic mirror 16a is used. The illumination NA of the illumination is NA ₁₀₂ (the NA of the light beam 102 in the figure), and NA ₁₀₂ = 1.42. Further, it is assumed that the distance from the pupil position 14a of the objective lens 14 to the position where the illumination light beam is reflected on the dichroic mirror 16a is 100 mm.

  In the case of using 488 nm laser light, the laser light is reflected by the dichroic mirror 16a and condensed at a position 14b on the pupil plane of the objective lens 14 (solid optical path 101). Then, the specimen 21 is collimated by the objective lens 14 to illuminate the specimen 21 (solid light path 102).

From this state, it is assumed that when the dichroic mirror 16a is switched to 17a (only the reflection surface is indicated by a dotted line), the angle of the reflection surface is shifted by 3 '. Then, the light reflected by the surface of the dichroic mirror 17a travels along the optical path 103 (dotted line), is condensed on 14b ′ shifted from the pupil position 14b of the objective lens 14, and illuminates the sample 21 by the optical path 104 (dotted line). At this time, the irradiation position deviation ΔR (position difference between 14b and 14b ′) at the pupil position 14a of the objective lens 14 is
ΔR = 100 × tan (3 ′ × 2) = 0.175 mm
Thus, the deviation amount ΔNA of the illumination NA at this time is as follows.

ΔNA = ΔR / fob = 0.175 / 3 = 0.58
Therefore, if the illumination NA is shifted in the direction of decreasing as shown in the optical path 104 (dotted line) in the figure,
NA ₁₀₄ = 1.42−0.058 = 1.362
Thus, the total reflection condition NA ≧ n ₂ (= 1.38) is not satisfied, and evanescent illumination is not achieved.

On the other hand, contrary to the illustrated optical paths 103 and 104 (dotted lines), when the illumination NA is increased by the same amount,
NA ₁₀₄ = 1.42 + 0.058 = 1.478
Thus, NAob (= 1.46) of the objective lens 14 is exceeded, and the condition of NAob (1.46) ≧ NA is not satisfied.

  In the above example, the angle difference of the excitation dichroic mirror is 3 ′. However, in a normal microscope, it may be shifted by 5 ′ or more, and the reflection position shift of the reflecting surface is also added.

  As described above, when the cube units 16 and 17 of the cube turret 15 are switched, the correction value corresponding to the cube unit 16 or 17 positioned on the optical path is read from the storage unit 27a, and the exit end of the fiber 7 is set to the pupil position of the objective lens 14. It can be seen that there is a need to perform control for accurately projecting to a position 14b offset from the center of 14a (position where the illumination NA is suitable for performing evanescent illumination).

  Therefore, in this way, when observation is performed using a plurality of fluorescent dyes, the excitation dichroic mirrors 16 and 17 having spectral characteristics matched to the respective fluorescent dyes are used in order to efficiently separate the excitation light and fluorescence. Are prepared, and the optimum ones of the excitation dichroic mirrors 16 and 17 are switched on the optical path for use. However, when the cube turret 15 is switched, the cube units 16 and 17 attached to the cube turret 15 are used. Even if there is an angle error or a folding position error in the dichroic mirrors 16a and 17a, and the projection position of the exit end face of the fiber 7 onto the pupil position 14a of the objective lens 14 is deviated, each cube unit 16 and 17 can be displaced. The output end face of the fiber 7 is moved to the electric stage 8b based on the correction value stored in the storage unit 27a every time. By performing the positioning control, it is possible to accurately project the emission end of the fiber 7 to a position 14b offset from the center of the pupil position 14a of the objective lens 14 (position where the illumination NA is suitable for performing evanescent illumination). . As a result, even if the excitation dichroic mirrors 16 and 17 having spectral characteristics matched to the fluorescent dye are switched and used, the illumination NA of the evanescent illumination can always be kept constant, and the total reflection fluorescence capable of stable vanescent illumination. Observation can be realized.

  Moreover, since the movement control of the exit end face of the fiber 7 using such a correction value can be automatically performed in conjunction with the cube turret switching, the illumination NA is shifted even when the cube turret 15 is switched. There is always no, and accurate evanescent illumination can always be performed.

  Furthermore, the cube turret 15 for switching the cube units 16 and 17 can be used as it is for an ordinary microscope, and it is not necessary to prepare a special one considering the switching accuracy. Since it can be handled only by replacing the light tube 8, it is possible to easily respond to the system upgrade from a normal microscope to one using evanescent illumination, and a high-performance microscope system can be realized.

  In the first embodiment described above, two types of cube units, the cube unit 16 for 488 nm excitation and the cube unit 17 for 543 nm excitation, are mounted on the cube turret 15, but the cube turret 15 can be switched. It is possible to attach different cube units to all four positions. In this case, the correction values obtained from the relationship between the four positions of the cube turret 15 and the cube units are stored in the storage unit 27a, Correction is performed by moving the electric stage 8b in conjunction with the switching of the turret 15. In this way, even if more cube units are used, accurate evanescent illumination can be performed.

Further, the angle error and the folding position error of each excitation dichroic mirror are a combination of an error caused by the assembly accuracy of the cube turret alone and a mechanical error of switching by the cube turret. In other words, although the cube turret has sufficient accuracy in the reproducibility of the stop position, an error occurs in the stop position between the cube mounting points due to the shaft misalignment of the rotation mechanism and the variation in the position accuracy of the positioning mechanism. Therefore, in consideration of these, the correction value of the electric stage 8b can be stored in the storage unit 27a in correspondence with the combination of the rotational position positions of the cube unit and the cube turret 15. This method is effective when the number of cube units to be used is increased and more cube units are used than the switching position of the cube turret 15, and correction values corresponding to the combination of the position to be mounted and the type of cube unit to be mounted are set. Since reading and correction can be performed, the illumination NA can always be kept accurate and accurate evanescent illumination can be realized even when more cube units are switched and replaced. Further, the correction value can be input in advance by a method of inputting at the time of shipment, or a correction value corresponding to when the user replaces the cube unit (not shown).
In this case, recognition means (for example, a sensor, a barcode, etc.) for recognizing the type of cube unit positioned on the optical path is provided. And if the cube unit arrange | positioned on an optical path is changed, the kind of cube unit after a change will be recognized by a recognition means, and the correction | amendment of the electric stage 8b will be performed with the correction value for every cube unit from this result.

  In this way, correction can be made even if the cube turret is rotated manually instead of electrically, and can be applied to an inexpensive microscope.

(Second Embodiment)
Next, a second embodiment of the present invention will be described.

  In the second embodiment, a laser beam is deflected by an optical scanning mirror in a total reflection fluorescence microscope system that performs evanescent illumination, spot light is scanned two-dimensionally on a sample, and fluorescence emitted from the sample is emitted from a confocal pin. A scanning unit for confocal observation through a hole is combined.

  FIG. 3 shows a schematic configuration of the second embodiment of the present invention, and the same parts as those in FIG.

  In this case, the laser light source unit 1 uses evanescent illumination and confocal observation in common, and the evanescent projector tube is replaced with a scanning unit 31.

  The emission end of the acoustooptic device 6 of the laser light source unit 1 is connected to the scanning unit 31 via the fiber 7.

  The scanning unit 31 is provided with a laser light introduction port 31 a for introducing laser light emitted from the fiber 7. A collimator lens 32 and an excitation dichroic mirror 33 are disposed on the optical path of the laser light emitted from the laser light introduction port 31a.

  The collimating lens 32 converts the laser light emitted from the laser light introducing port 31a into collimated light. The excitation dichroic mirror 33 has a characteristic that reflects the wavelength (488 nm, 543 nm) of the laser beam and transmits the wavelength region of the fluorescence emitted from the sample 21.

  On the reflection optical path of the excitation dichroic mirror 33, an optical deflection mirror unit 34 as optical scanning means is arranged. The light deflection mirror unit 34 has two sets of galvano scanner mirrors that deflect light in the vertical direction of the paper surface and the direction perpendicular to the paper surface. The galvano scanner mirrors scan the laser light in a two-dimensional direction. Yes.

  A pupil projection lens 35 is disposed on the optical path of the laser light emitted from the light deflection mirror unit 34. A condensing lens 36 is disposed on the optical path of the laser light emitted from the pupil projection lens 35. The condenser lens 36 converts the laser light emitted from the pupil projection lens 35 into parallel light (an optical path 36a indicated by a dotted line), and attaches it to a cube unit (not shown) in which only a reflection mirror attached to the cube turret 15 is attached. Incident light is incident.

  Reference numeral 38 denotes a lens unit for evanescent illumination, which is inserted into the optical path only during evanescent illumination, and is removed from the optical path in confocal observation. That is, in the evanescent illumination, it is necessary to focus the laser beam on the pupil position 14a of the objective lens 14. For this reason, the imaging position 37 that is collected by the pupil projection lens 35 is connected to the pupil position 14a of the objective lens 14. The lens unit 38 can be inserted into the optical path for imaging.

  On the other hand, on the transmission optical path of the excitation dichroic mirror 33, a reflection mirror 39 constituting a confocal observation means, a confocal lens 40, a confocal pinhole 41, and a barrier filter for extracting a fluorescence wavelength region to be detected by cutting the excitation laser light. 42 and a photodetector 43 are arranged. For example, a photomultiplier is used as the photodetector 43 here.

  In such a configuration, first, confocal observation will be described.

  In this case, the laser light from the laser light source unit 1 is emitted from the acoustooptic device 6 and introduced into the laser light introduction port 31 a of the scanning unit 31 through the fiber 7. Then, it is converted into parallel light by the collimating lens 32, reflected downward as excitation laser light by the excitation dichroic mirror 33, and enters the light deflection mirror unit 34.

  The light deflection mirror unit 34 deflects laser light by two sets of galvano scanner mirrors that deflect light in a vertical direction on the paper surface and a direction perpendicular to the paper surface. The laser light deflected by the light deflection mirror unit 34 passes through the pupil projection lens 35 and is converted into parallel light by the condenser lens 36 (optical path 36a indicated by a dotted line). At this time, the lens unit 38 for evanescent illumination is removed from the optical path.

  The laser light converted into parallel light by the condenser lens 36 is reflected upward by a cube unit (not shown) having only a reflection mirror attached to the cube turret 15, and is incident on the objective lens 14 as parallel light. The light is condensed by the objective lens 14 and a light spot is formed on the sample 21. This light spot is scanned two-dimensionally on the sample 21 by the light deflection of the light deflection mirror unit 34.

  When fluorescence is emitted by two-dimensional scanning of the light spot on the sample 21, this fluorescence travels in the opposite direction to the excitation laser light, and is excited through the condenser lens 36, pupil projection lens 35, and light deflection mirror unit 34. The dichroic mirror 33 is reached. The fluorescence that has reached the excitation dichroic mirror 33 is transmitted through the excitation dichroic mirror 33 having the characteristics that the laser wavelength is reflected and the fluorescence wavelength is transmitted, is reflected by the reflection mirror 39, is confocal lens 40, confocal pinhole 41, barrier. The light passes through the filter 42 and is detected by the photodetector 43.

  Next, fluorescence observation using evanescent illumination will be described.

  In the above-mentioned confocal observation, it is necessary to make the laser light incident on the objective lens 14 as parallel light, whereas in the evanescent illumination, it is necessary to focus the laser light on the pupil position 14a of the objective lens 14. For this purpose, a lens unit 38 for forming an imaging position 37 condensed by the pupil projection lens 35 at the pupil position 14a of the objective lens 14 is inserted into the optical path.

  In addition, in order to control the position at which the image is formed on the pupil position 14a of the objective lens 14 (deciding the illumination NA), one galvano scanner mirror (on the paper surface) in the light deflection mirror unit 34 composed of two sets of galvano scanner mirrors. An objective lens that is optically conjugate with the imaging position 37 by changing the imaging position 37 of the pupil projection lens 35 by deflecting the galvano scanner mirror. This is done by changing the light collection position at the 14 pupil positions 14a.

  Also in this case, the control unit 27 reads out the correction value corresponding to the rotational position of the cube turret 15 to which the cube units 16 and 17 are mounted from the storage unit 27a, and uses this correction value to detect the galvano of the light deflection mirror unit 34. By controlling the deflection angle of the scanner mirror and changing the imaging position 37 by the pupil projection lens 35, the condensing position at the pupil position 14a of the objective lens 14 optically conjugate with the imaging position 37 is changed. Let

  From this state, the laser light for evanescent illumination from the laser light source unit 1 is transmitted from the pupil projection lens 35 through the lens unit 38 and the condenser lens 36 as shown by the solid line optical path 36b in the drawing, and the cube unit 16 (or 17). ), Is reflected toward the objective lens 14 side, is condensed at a position 14b offset from the optical axis center of the pupil position 14a of the objective lens 14, and evanescent illumination is performed on the sample 21 via the objective lens 14.

  In this case, as described above, in the fluorescence observation using the fluorescent dye Fitc of 488 nm as the excitation laser wavelength, the cube turret 15 is rotationally switched by the motor 18 and the cube unit 16 is arranged on the optical path. In fluorescence observation using the 543 nm fluorescent dye Tritc, the cube turret 15 is rotationally switched by the motor 18 to place the cube unit 17 on the optical path. Further, the fluorescence emitted from the sample 21 by the evanescent illumination is observed by the CCD camera 24 or the visual observation unit 26 as in the first embodiment described above.

  Accordingly, when the cube turret 15 is switched, the dichroic mirrors 16a and 17a of the cube units 16 and 17 attached to the cube turret 15 have an angle error and a folding position error. Correction stored in the storage unit 27a for each of the cube units 16 and 17 even if there is a deviation in the condensing position at the pupil position 14a of the objective lens 14 that is optically conjugate with the imaging position 37 by the lens 35. By controlling the deflection angle of the galvano scanner mirror of the light deflection mirror unit 34 based on the value, the optical axis center of the pupil position 14a of the objective lens 14 that is optically conjugate with the imaging position 37 by the pupil projection lens 35 The laser beam can be accurately collected at the position 14b offset from the position. As a result, even if the excitation dichroic mirrors 16 and 17 having spectral characteristics matched to the fluorescent dye are switched and used, the illumination NA of the evanescent illumination can always be kept constant, and the total reflection fluorescence capable of stable vanescent illumination. Observation can be realized.

  Since the deflection control of the galvano scanner mirror of the light deflection mirror unit 34 using such a correction value can be automatically performed in conjunction with the cube turret switching, the NA of the illumination is maintained even when the cube turret 15 is switched. There is no deviation at all and accurate evanescent illumination can always be performed.

  Further, a light deflection mirror unit 34 (including a control driver (not shown)) for performing confocal observation with a scanning laser microscope is controlled for correcting the condensing position of the laser light at the pupil position 14a of the objective lens 14. Since it can be shared with functions, the system can be miniaturized and the price can be reduced. Since the laser light source for confocal observation and the laser light source for evanescent illumination can be shared, the system can be further reduced in size and cost. Also in this case, simply replacing the epi-illumination projection tube with the scanning unit 31 makes it possible to easily respond to the system upgrade from a normal microscope to one using evanescent illumination. A system can be realized.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.

The figure which shows schematic structure of the 1st Embodiment of this invention. The figure which expands and shows FIG. 1 in order to demonstrate in detail the evanescent illumination of 1st Embodiment. The figure which shows schematic structure of the 1st Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Laser light source unit, 2 ... Argon laser 3 ... Green helium neon laser, 4 ... Reflection mirror 5 ... Dichroic mirror, 6 ... Acousto-optic element 7 ... Fiber, 8 ... Evanescent projector tube 8a ... Laser introduction part, 8b ... Electricity Stage, 8d ... Arrow 801 ... Projection tube body, 802 ... Light guide tube, 9 ... Inverted microscope body 9a ... Transmission light path, 9b ... Reflection light path, 9c ... Visual observation light path 10 ... Lens, 11 ... Reflection mirror, 12 ... Epi-illumination Illumination light source 13 ... Condensing lens, 14 ... Objective lens, 14a ... Pupil position 14b, 14b '... Position, 15 ... Cube turret, 16.17 ... Cube unit 16a ... Excitation dichroic mirror, 16b ... Barrier filter 17 ... Cube unit 17a ... excitation dichroic mirror 17b ... barrier filter, 18 ... motor 19 ... Material stage 20 ... Cover glass 21 ... Sample, 22 ... Imaging lens, 23 ... Reflection mirror 24 ... CCD camera, 25 ... Reflection mirror, 26 ... Visual observation unit 27 ... Control unit, 27a ... Storage unit 31 ... Scanning unit 31a ... Laser light introduction port 32 ... Collimate lens 33 ... Excitation dichroic mirror 34 ... Light deflection mirror unit 35 ... Pupil projection lens 36 ... Condensing lens 36a, 36b ... Optical path 37 ... Image forming position 38 ... Lens unit , 39: reflection mirror 40 ... confocal lens, 41 ... confocal pinhole 42 ... barrier filter, 43 ... photodetectors 101-104 ... optical path

Claims (6)

A light source that generates laser light of a plurality of wavelengths;
A condensing optical system for generating evanescent illumination by condensing the laser light from the light source on a plane perpendicular to the optical axis including the pupil of the objective lens;
An observation optical system for observing fluorescence generated from the sample by the evanescent illumination;
A plurality of types of wavelength-selective optical elements that are provided so as to be switched or exchangeable for each wavelength of the laser light of the light source, reflect the laser light, and transmit fluorescence from the sample;
A microscope system comprising: a correcting unit that corrects a deviation of a condensing position of the laser light reflected by each of the plurality of wavelength selection optical elements at the objective lens. The correction unit includes a storage unit that stores a correction value according to an error related to the wavelength selection optical element, and is based on the correction value stored in the storage unit by switching or replacement of the wavelength selection optical element. The microscope system according to claim 1, wherein a deviation of a condensing position of the laser light from the light source at the objective lens is corrected. A fiber for deriving laser light from the light source;
The condensing optical system projects the laser beam emission end of the fiber onto a plane perpendicular to the optical axis including the pupil of the objective lens,
The microscope system according to claim 2, wherein the correction unit includes a moving unit that horizontally moves a laser beam emission end of the fiber. A deflection mirror that deflects the laser light from the light source, and further having a confocal observation function that two-dimensionally scans the sample with a light spot by deflecting the deflection mirror;
The condensing optical system condenses the laser beam deflected by the deflecting mirror onto a surface perpendicular to the optical axis including the first state in which spot illumination is performed on the sample and the pupil of the objective lens. The state can be switched,
The microscope system according to claim 2, wherein the correction unit is configured to control a deflection angle of the deflection mirror in the second state. 5. The wavelength selective optical element includes an excitation dichroic mirror mounted on a cube unit, and the excitation dichroic mirror can be switched by a cube turret equipped with the cube unit. The microscope system according to any one of the above. The microscope system according to claim 5, wherein the correction unit determines a correction value of the storage unit from a relationship between a position of the cube unit and the cube turret to which the cube unit is mounted.

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