JP4700334B2 - Total reflection fluorescence microscope - Google Patents

Description

  The present invention relates to a total reflection fluorescent microscope.

  Recently, functional analysis of living cells has been actively performed. In the functional analysis of these cells, in particular, to observe the function of the cell membrane, total reflection fluorescence images from the cell membrane and its vicinity are used. The total reflection fluorescence microscope (TIRFM) to be obtained has attracted attention.

  This total reflection fluorescent microscope uses a light called evanescent light that oozes in a slight range of several hundred nm or less on the specimen side when the illumination light is totally reflected at the interface between the cover glass and the specimen. Since only a small range of fluorescence near the cover glass is observed by the excitation method, the background is very dark and high-contrast fluorescence observation or weak fluorescence observation is possible.

  By the way, in the field of biological research using such a total reflection fluorescent microscope, there is a case where it is desired to observe a shallower surface near the boundary surface with good contrast, and the illumination light reaches a certain depth. Sometimes you want to observe a wide range. For this reason, it is desirable that the penetration depth of the evanescent light can be changed according to the observation target.

  The penetration depth of evanescent light from the boundary surface is disclosed in Non-Patent Document 1, and it is known that the following formula holds.

d = λ / 4π {(n ₁ ² sin θ ₁ ² −n ₂ ² )} ^1/2 (1)
Here, d: penetration depth of evanescent light, λ: wavelength of light, n ₁ : incident side (cover glass) refractive index, θ ₁ : incident angle, n ₂ : outgoing side (sample) refractive index. .
Therefore, as is apparent from the above equation, the greater the incident angle of the illumination light with respect to the boundary surface of the total reflection illumination angle, that is, the inclination angle of the illumination light with respect to the normal of the boundary surface, the shallower the penetration depth of the evanescent light. Become.

As disclosed in Patent Document 1, the position of the focal point of the condensed light in the rear focal plane of the objective lens is adjusted by the rotation of the reflecting optical system, as disclosed in Patent Document 1, so that the specimen is used. There is one in which the incident angle of light introduced to the side can be adjusted.
JP 2002-31762 A Daniel Axelrod, “5. Total Internal Reflection Fluorescence at Biological Surfaces”, Noninvasive Technics in Cell Biology: 93-127, 1990, Wiley-Liss, Inc., pp111-113

  However, as shown in FIG. 2, since the pupil diameter and the pupil position differ depending on the type of the objective lens, the illumination spot deviates from the pupil position of the objective lens when the objective lens is switched. For example, the pupil position of the objective lens 18 with 100 × magnification ((b) in FIG. 2) is closer to the sample plane 21 than the pupil position of the objective lens 18 with 60 × magnification ((a) in FIG. 2). It has become. For this reason, the position of the illumination spot must be adjusted so that the illumination spot is correctly connected to this pupil position.

  Even when the objective lens is not switched, the position of the illumination spot may shift due to chromatic aberration of the optical system when the wavelength of the laser light is changed. Therefore, even if this is not corrected, correct evanescent illumination cannot be obtained.

  As described above, in the total reflection illumination microscope, when the objective lens is switched, it is necessary to change the condensing position of the illumination light always parallel to the specimen due to the difference in the characteristic (pupil position) of the objective lens. For this reason, when the objective lens is switched, the optical path length between the light source and the objective lens must be changed.

  When the objective lens is switched, the illumination angle of the illumination light may change due to the difference in characteristics (magnification) of the objective lens. In order to obtain the exudation light having the same depth, it is necessary to make it the same as before changing the irradiation angle of the illumination light.

  An object of this invention is to provide the total reflection fluorescence microscope which can always irradiate a sample with parallel light.

  In the present invention, when changing the magnification of the objective lens, the position of the illumination spot is adjusted in the direction perpendicular to the optical axis in accordance with the pupil diameter. In addition, when the pupil position of the objective lens or the position of the illumination spot changes, the position of the illumination spot is adjusted in the optical axis direction. Specifically, it is as follows.

The invention according to aspect of the present invention includes a light source, a lighting angle control means for controlling the irradiation angle of irradiating illumination light to the specimen from the light source, an objective lens switching means for switching a plurality of objective lenses having different magnifications, wherein Optical path length variable control means for changing the focusing position in the optical axis direction for condensing illumination light in the vicinity of the pupil position of the objective lens, and the irradiation angle control means and optical path length for each objective lens with different magnification and / or pupil position Storage means for storing the adjustment value of the variable control means, and control means for controlling the irradiation angle control means and / or the optical path length variable control means using the adjustment value when the objective lens is switched; It is characterized by comprising. In addition, it is preferable that the irradiation angle control unit adjusts, for example, when the wavelength of the irradiation light from the light source is changed or when the irradiation angle is changed.

  According to the present invention, the specimen can always be irradiated with parallel light.

Embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a diagram showing a schematic configuration of a total reflection fluorescence microscope according to an embodiment of the present invention.
In this embodiment, fluorescence observation by evanescent illumination, laser light is deflected by an optical scanning mirror, spot light is two-dimensionally scanned on the specimen, and fluorescence emitted from the specimen is confocally observed through a confocal pinhole. A configuration that can do both is shown. This scanning mirror is used to two-dimensionally scan the spot light on the specimen during confocal observation, and is used to vary the illumination angle that determines the penetration depth during evanescent illumination.

  In FIG. 1, a laser light source unit 1 includes an argon (Ar) laser 2 that oscillates, for example, 488 nm laser light and a green helium neon laser 3 that oscillates 543 nm laser light, which are used for evanescent illumination.

  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 laser beams of 488 nm and 543 nm, and enables switching of the respective wavelengths. As another wavelength selection means, a plurality of LD light sources that emit laser beams of different wavelengths are arranged in place of the acousto-optic element 6, and a current value supplied to the power source of these LD light sources is varied and turned on / off. A laser beam having a wavelength to be selected may be selected. In place of the acousto-optic element, an electro-optic element or a liquid crystal shutter may be substituted.

  The emitting end of the acoustooptic device 6 of the laser light source unit 1 is connected to the scanning unit 41 via the fiber 7. The scanning unit 41 is provided with a laser light introduction port 41 a for introducing laser light emitted from the fiber 7. A collimator lens 42 and an excitation dichroic mirror 43 are disposed on the optical path of the laser light emitted from the laser light introduction port 41a.

  The collimating lens 42 converts laser light emitted from the laser light introduction port 41a into collimated light. The excitation dichroic mirror 43 has a characteristic that reflects the wavelength (488 nm, 543 nm) of the laser light and transmits the wavelength region of the fluorescence emitted from the specimen 19.

  On the reflection optical path of the excitation dichroic mirror 43, an optical deflection mirror unit 44 as an optical scanning unit is arranged. The light deflection mirror unit 44 has two sets of galvano scanner mirrors that deflect light in the vertical direction of the paper surface and in a direction perpendicular to the paper surface. When confocal observation is performed using these galvano scanner mirrors, the spot light is two-dimensional on the sample surface. During scanning and evanescent illumination, the angle of illumination of the specimen is varied.

  A pupil projection lens 45 is disposed on the optical path of the laser light emitted from the light deflection mirror unit 44. A condensing lens 46 is disposed on the optical path of the laser light emitted from the pupil projection lens 45. The condenser lens 46 converts the laser light emitted from the pupil projection lens 45 into parallel light (an optical path indicated by a dotted line) so as to enter the cube turret 14. Then, the light is reflected upward by the cube unit 16 including the total reflection mirror 16 a attached to the cube turret 14, and spot light is connected to the sample 19 by the objective lens 18. In this way, the spot light is connected to the specimen 19 for illumination during confocal observation.

  The prism 10 is disposed between the pupil projection lens 45 and the condenser lens 46 so as to be detachable. The prism 10 is a prism for evanescent illumination, 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 18a of the objective lens 18. For this reason, the imaging position 45a collected by the pupil projection lens 45 is connected to the pupil position 18a of the objective lens 18. The prism 10 can be inserted into the optical path for imaging. Since the imaging position at the pupil position 18a of the objective lens 18 changes in the radial direction of the objective lens 18 due to the deflection of the light deflection mirror unit 44, the irradiation angle to the specimen can be varied.

The prism 10 can be inserted into and removed from the optical path by moving in the direction indicated by the arrow by a motor (not shown) driven by an instruction from the control unit 29. When the prism 10 is inserted on the optical path, the laser light is reflected by the prism 10, reflected again by the prism 10 via the relay lens 11, the prism 48 and the relay lens 12, and enters the condenser lens 46 ( The optical path shown by the solid line). Here, the prism 48 can be moved in the direction of the arrow in the figure by a motor 49 that is driven by the control of the control unit 29. By this movement, the condensing position for condensing light on the pupil position 18a of the objective lens 18 is changed in the optical axis direction. In other words, the optical path length can be changed.

  On the other hand, on the transmission optical path of the excitation dichroic mirror 43, a reflection mirror 50, a confocal lens 51, a confocal pinhole 52, and a barrier filter that extracts the fluorescence wavelength region to be detected by cutting the excitation laser light. 53 and a photodetector 54 are arranged. For example, a photomultiplier is used for the photodetector 54 here.

  The cube turret 14 is disposed in the optical path of the laser light emitted from the condenser lens 46. The cube turret 14 can hold a plurality of cube units, and can be rotated and switched by a motor 15 via a sliding mechanism such as a bearing (not shown). In the illustrated example, two types of cube units 16 and 47 are mounted, and when performing total reflection fluorescence observation, the cube unit 47 is positioned on the optical path of the laser light from the condenser lens 46.

  The cube unit 47 is a wavelength selection optical element that reflects light having wavelengths of 488 nm and 543 nm as excitation light, and transmits fluorescence having wavelengths of 500 to 540 nm and 560 to 600 nm generated from the specimen 19 by the excitation wavelength. Excitation dichroic mirror 16a having

  The objective lens 18 is disposed in the reflected light path of the dichroic mirror 47a (positioned on the optical path of the laser light from the condenser lens 46) of the cube unit 47. In the present embodiment, there are a plurality of objective lenses 18 (only one is shown in the figure), and a desired objective lens can be switched by the motor 17 driven by the control of the control unit 29.

  A specimen 19 stained with a fluorescent dye is disposed at the focal position of the objective lens 18. The specimen 19 is disposed on the stage 20 of the inverted microscope body 9 and is fixed to the cover glass 21. In this case, the cover glass 21 and the objective lens 18 are filled with immersion oil for ensuring a high NA and causing total reflection by evanescent illumination.

  An imaging lens 22 and a reflection mirror 23 constituting an observation optical system are arranged in a transmission optical path 22a of a dichroic mirror 47a (positioned on the optical path of laser light from the condenser lens 46) of the cube unit 47. Yes.

  The reflection mirror 23 can be inserted and removed from the optical path by a switching mechanism (not shown). 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 filter wheel 24 constituting a fluorescence observation unit is disposed in the reflection optical path of the reflection mirror 23. The filter wheel 24 holds a plurality of (two in the illustrated example) filters 24a and 24b, and can be switched to the optical path by a motor 25 via a sliding mechanism (not shown). In this case, the filter 24a transmits fluorescence generated at an excitation wavelength of 488 nm, and the filter 24b transmits fluorescence generated at an excitation wavelength of 543 nm, for example. In the illustrated example, the filter 24a is positioned on the optical path.

  On the optical path that has passed through the filter wheel 24, a CCD camera 26 as an imaging means is disposed. The fluorescence from the specimen 19 that has passed through the imaging lens 22 is guided to the CCD camera 26 and imaged on the imaging surface 26a.

  In a state where the reflection mirror 23 is out of the optical path, the reflection mirror 27 and the visual observation unit 28 are disposed in the visual observation optical path 22b.

  On the other hand, the acousto-optic element 6, the light deflection mirror unit 44, the motor 15 for rotationally driving the cube turret 14, the motor 25 for rotationally driving the filter wheel 24, the CCD camera 26, and the driving motor 49 for the prism 48 are used as control means. The control unit 29 is connected. The control unit 29 outputs control signals to the acoustooptic device 6, the light deflection mirror unit 44, the motors 15, 25, and 49 and the CCD camera 26, and processes image data captured by the CCD camera 26 to the monitor 30. It can be displayed. In addition, the control unit 29 is provided with a storage unit 29a as a storage unit.

  In this case, the storage unit 29a has the light necessary for obtaining the same irradiation angle at the position of the prism 48 that changes the optical path length and the magnification of the objective lens in order to align the position of the illumination spot with the pupil position of each objective lens. The deflection angle of the deflection mirror unit 44 is stored. Thereby, even when the objective lens is switched, the position of the prism 48 for aligning the position of the illumination spot with the pupil position of the objective lens and the deflection angle of the light deflection mirror unit 44 are stored, so that the illumination is performed immediately. The spot can be adjusted to the pupil position of the objective lens. As a result, even when switching to an objective lens having a different pupil position or magnification, the seepage depth can be made constant, and the specimen can always be irradiated with parallel light, so that accurate total reflection illumination can be performed.

  Even if the objective lens is not switched, when the wavelength of the laser light from the light source is changed, the position of the illumination spot is different from the pupil position of the objective lens with respect to the optical axis direction due to chromatic aberration in the optical axis direction of the optical system. There may be deviation. Also in this case, the position of the prism 48 in which the position of the illumination spot matches the pupil position of the objective lens is stored in the storage unit 29a for each wavelength. As a result, even if the illumination light condensed on the pupil surface of the objective lens has chromatic aberration in the optical axis direction, the position of the illumination spot comes to the pupil position of the objective lens. Therefore, accurate total reflection illumination can be performed.

  In addition, when selecting a wavelength from a plurality of wavelengths and sequentially irradiating the sample while switching the wavelengths and acquiring fluorescence images with a plurality of excitation wavelengths in a time division manner, the sample is applied to the sample at an irradiation angle set for each wavelength. Control is performed so that the control value of the irradiation angle control means (deflection angle of the light deflection mirror unit 44) and the control value of the optical path length variable control means (position of the prism 48) are sequentially switched in synchronization with wavelength switching so as to irradiate. It is preferable to do. Thereby, even if there is chromatic aberration in the optical axis direction, a sequential image of multiple stained samples at each wavelength at a desired (constant) penetration depth can be obtained accurately and extremely easily.

  Further, as the positional deviation of the illumination spot other than when the objective lens is switched, it is conceivable that the illumination light is focused on the vicinity of the pupil plane of the objective lens when the irradiation angle is changed. Also in this case, the position of the prism 48 such that the position of the illumination spot for each illumination angle (the deflection angle of the light deflection mirror unit 44) matches the pupil position of the objective lens is stored in the storage unit 29a. As a result, even when the irradiation angle is changed, the position of the illumination spot comes to the pupil position of the objective lens, so that the specimen can always be irradiated with parallel light when the irradiation angle is switched. Total reflection illumination can be performed.

  In the above embodiment, the means for moving the condensing position in the optical axis direction is described as “optical path length varying means” (in the embodiment, a movable prism). If a method of changing the optical path length is used, it is preferable to adjust only the condensing position and not to change other optical parameters, and it is preferable. However, a method of moving the lens or adjusting the optical axis direction of the condensing position using a zoom optical system is also possible.

  According to the embodiment of the present invention, since the optical path length is adjusted according to the switching of the objective lens, the switching of the wavelength, and the change of the irradiation angle, the specimen can always be irradiated with the parallel light. Total reflection illumination can be performed.

  The present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the scope of the invention at the stage of implementation. In the above-described embodiment, the positional deviation of the illumination spot has been described only by switching the objective lens, switching the wavelength, and changing the irradiation angle. However, the present invention is not limited to this, and the position of the illumination spot is shifted in the optical axis direction. Anything can be applied. Furthermore, in the above description, objective lens switching, wavelength switching, and correction for irradiation angle change have been described separately, but they may be applied at the same time, or may be applied in appropriate combination. Further, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements.

  In addition, for example, even if some structural requirements are deleted from all the structural requirements shown in each embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and the effect described in the effect of the invention Can be obtained as an invention.

The figure which shows schematic structure of the total reflection fluorescence microscope concerning one embodiment of this invention. The figure for demonstrating the position shift of the illumination spot when an objective lens is switched.

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, 9 ... Inverted microscope main body, 10 ... Prism, 11, DESCRIPTION OF SYMBOLS 12 ... Relay lens, 14 ... Cube turret, 15 ... Motor, 16 ... Cube unit, 16a ... Dichroic mirror, 17 ... Motor, 18 ... Objective lens, 18a ... Pupil position, 19 ... Sample, 20 ... Stage, 21 ... Cover glass , 22 ... imaging lens, 22a ... transmission optical path, 22b ... visual observation optical path, 23 ... reflection mirror, 24 ... filter wheel, 24a, 24b ... filter, 25 ... motor, 26 ... CCD camera, 26a ... imaging surface, 27 ... Reflection mirror, 28 ... unit for visual observation, 29 ... control unit, 29a ... storage unit, 30 ... monitor DESCRIPTION OF SYMBOLS 41 ... Scanning unit, 41a ... Laser beam introduction port, 42 ... Collimating lens, 43 ... Excitation dichroic mirror, 44 ... Light deflection mirror unit, 45 ... Pupil projection lens, 45a ... Imaging position, 46 ... Condensing lens, 47 ... Cube unit, 47a ... dichroic mirror, 48 ... prism, 49 ... motor, 50 ... reflection mirror, 51 ... confocal lens, 52 ... confocal pinhole, 53 ... barrier filter, 54 ... photodetector.

Claims (10)

A light source;
An irradiation angle control means for controlling an irradiation angle at which the specimen is irradiated with illumination light from the light source;
Objective lens switching means for switching a plurality of objective lenses having different magnifications;
An optical path length variable control means for varying the light condensing position in the optical axis direction where the illumination light is condensed near the pupil position of the objective lens;
Storage means for storing adjustment values of the irradiation angle control means and the optical path length variable control means for each objective lens having different magnification and / or pupil position;
And a control means for controlling the irradiation angle control means and / or the optical path length variable control means using the adjustment value when the objective lens is switched. A light source;
An optical path length variable control means for varying in the optical axis direction a condensing position where the illumination light from the light source is condensed near the pupil position of the objective lens;
Storage means for storing an adjustment value of the optical path length variable control means for each wavelength of the illumination light;
A total reflection fluorescent microscope comprising: a control unit that adjusts the optical path length variable control unit using the adjustment value when the wavelength of the illumination light is switched. The control means performs wavelength selection from a plurality of wavelengths, sequentially irradiates the sample while switching the wavelengths, and acquires a fluorescence image with a plurality of wavelengths of illumination light in a time division manner, and sets an optical path length set for each wavelength. 3. The total reflection fluorescence microscope according to claim 2, wherein the adjustment value of the optical path length variable control means is sequentially switched in synchronization with the wavelength switching so that the sample is irradiated with the lens. An irradiation angle control means for controlling an irradiation angle for irradiating the specimen with illumination light from the control means;
Said control means, so as to irradiate the specimen by the irradiation angle is set for each wavelength of the illumination light, according to claim 2, wherein sequentially switching the control values of the irradiation angle control means in synchronism with the wavelength switching Total reflection fluorescence microscope. A light source capable of oscillating at least one wavelength;
An irradiation angle control means for controlling an irradiation angle at which the specimen is irradiated with illumination light from the light source;
An optical path length variable control means for varying the light condensing position in the optical axis direction where the illumination light is condensed near the pupil position of the objective lens;
Storage means for storing adjustment values of the irradiation angle control means and the optical path length variable control means for each objective lens having different magnification and / or pupil position;
A total reflection fluorescent microscope characterized by comprising control means for controlling the irradiation angle control means and / or the optical path length variable control means in conjunction with switching of the objective lens using the adjustment value stored in the storage means. . Wavelength selection means for selecting the wavelength of illumination light from a light source that oscillates at least two wavelengths;
The storage means stores, for each wavelength, a correction value of the optical axis direction condensing position shift near the pupil position of the objective lens caused by chromatic aberration in the optical axis direction,
6. The total reflection fluorescent microscope according to claim 5 , wherein the control means controls the optical path length variable control means in conjunction with the switching of the wavelength of the illumination light using the correction value for each wavelength. The storage means stores an irradiation angle control value for each wavelength of illumination light,
The control means uses the irradiation angle control value for each wavelength to control the irradiation angle control means in conjunction with the switching of the wavelength of the illumination light, so that the amount of exudation of evanescent illumination is independent of the wavelength. 6. The total reflection fluorescent microscope according to claim 5 , wherein the total reflection fluorescence microscope is set to a constant value or a desired value for each wavelength. The total reflection fluorescence microscope according to claim 6 or 7 , wherein the total reflection fluorescence microscope acquires a fluorescence image for each wavelength of a plurality of illumination lights in a time division manner. The storage means stores, for each irradiation angle, a correction value for correcting a condensing position shift in the optical axis direction caused by a field curvature of illumination light condensed near the pupil plane of the objective lens,
6. The total reflection fluorescent microscope according to claim 5 , wherein the control means controls the optical path length variable control means in conjunction with the change of the irradiation angle by using the correction value for each irradiation angle. A light source;
Objective lens switching means for switching a plurality of objective lenses having different pupil positions;
An optical path length variable control means for varying in the optical axis direction a condensing position where the illumination light from the light source is condensed near the pupil position of the objective lens;
Storage means for storing an adjustment value of the optical path length variable control means for each objective lens having a different pupil position;
Control means for controlling the optical path length variable control means using the adjustment value stored in the storage means when the objective lens is switched;
A total reflection fluorescent microscope characterized in that the illumination light from the light source is condensed near the pupil position for each objective lens having a different pupil position.

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