JP2009042411A - Illuminator for microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an illuminator for a microscope, wherein spot illumination with high accuracy is achieved. <P>SOLUTION: A laser beam emitted from the emitting part 12B of a single mode fiber 12 is formed into a parallel luminous flux by a collimator lens 61, then transmitted to a diaphragm 62, once condensed by a relay lens 14, and formed into a parallel luminous flux again by a field lens 16. The laser beam formed in the parallel luminous flux is reflected in the direction of the optical axis of an objective lens 18 by a dichroic mirror 17, and enters within the range of the image side focal face (pupil face) BF of the objective lens 18, which satisfies conditions for total reflection on the specimen 2 side face of a cover glass 5. Then, the laser beam condenses on the contact face of the specimen 2 with the cover glass 5 by the objective lens 18, and spot-illuminates the specimen 2. This invention can be used in, for example, the illuminator for the microscope. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an illumination device for a microscope, and more particularly to an illumination device for a microscope that performs total reflection illumination.

  In recent years, in the study of cell behavior, the movement of intracellular substances (mainly proteins) has been tracked. By tracking the movement of intracellular substances, major cellular activities such as communication inside and outside the cell, cell division, and cell migration are elucidated, leading to the elucidation of biological functions and diseases. Therefore, in order to track the movement of the intracellular substance, a fluorescent substance having a property of emitting fluorescence when irradiated with light of a predetermined wavelength is attached to the intracellular substance to be observed as a fluorescent label and observed with a microscope (hereinafter referred to as fluorescence). It is generally performed (referred to as Patent Document 1).

  Recently, fluorescent substances such as PA-GFP (Photoactivatable GFP), Kaede (trademark), and Dronpa (trademark) have been developed and commercialized, and the degree of freedom of tracking intracellular substances has been improved. PA-GFP is a fluorescent substance that changes from a state that does not emit fluorescence to a state that emits fluorescence when irradiated with light of a specific wavelength. Kaede (trademark) is a fluorescent substance that changes from green to red fluorescence when irradiated with light of a specific wavelength. Dronpa (trademark) changes from a state that does not emit fluorescence by irradiating light of a specific wavelength, and then changes to a state that does not emit fluorescence by irradiating light of another wavelength. It is a fluorescent substance that can be repeatedly returned.

  In fluorescence observation, in addition to light stimulation (PA (Photo Activation) or Photo Conversion) that stimulates the specimen with light as described above, FRAP (Fluorescence Recovery after Photobleaching), FCS (Fluorescence Correlation Spectroscopy) ) Is generally used.

  In the fluorescence observation method using light stimulation, for example, the fluorescence label of an arbitrary substance is irradiated with light of a predetermined wavelength at an arbitrary timing, thereby controlling the on / off of the fluorescence and tracking the intracellular substance. .

  In the fluorescence observation method using FRAP, for example, by using a fading that causes the fluorescent label to cease to emit fluorescence due to the irradiation energy by irradiating light of a predetermined wavelength stronger than a predetermined intensity, the fluorescence of the fluorescent label is partially Is erased, and the fluorescent substance moving to that part is observed.

  In the fluorescence observation method using FCS, for example, the irradiation range of illumination light is limited to a very small volume of several fl (ferm liter), and the fluorescent signal change in that region is recorded and analyzed to attach a fluorescent label. The behavior of the substance is clarified (for example, see Patent Document 2).

  By the way, in recent years, as one of the fluorescence observation methods, total reflection illumination fluorescence that performs fluorescence observation of a sample using illumination using total reflection (hereinafter referred to as total reflection illumination or TIRF (Total Internal Reflection Fluorescence)). Observation methods are becoming popular. In conventional total reflection illumination, illumination is performed within a range (hereinafter also referred to as a TIRF range) that satisfies the condition of total reflection on the specimen side surface of the cover glass that protects the specimen on the image side focal plane (pupil plane) of the objective lens. By condensing the light and irradiating the sample with parallel light, it is possible to uniformly illuminate a predetermined range of the sample (see, for example, Patent Document 3). For example, when an objective lens having a magnification of 100 is used, the inside of a circle with a diameter of 0.1 mm of the sample is illuminated. Compared with the conventional epifluorescence observation method, the total reflection illumination fluorescence observation method is limited to a very shallow range in which the illumination light is irradiated in the depth direction of the specimen, and is not affected by the background. It becomes possible to obtain information near the surface of the specimen with high sensitivity.

JP 2006-3662 A Special table 2003-524180 gazette Japanese Patent Laid-Open No. 2004-85796

  Conventionally, when observing a fine region of a specimen by fluorescence observation using a technique such as light stimulation, partial fading, and FSC, spot illumination that collects illumination light on the specimen is used. The distribution of the spot illumination area is calculated using the numerical aperture (NA) of the objective lens, assuming that the specimen is homogeneous throughout the area through which the illumination light passes. Since both have non-homogeneous and complex structures, the refractive index, light scattering state, and the like vary depending on the position and direction of the specimen. Therefore, it is very difficult to form a spot for illumination inside the specimen as calculated, and the illuminated area is distorted from an ideal symmetrical shape. Therefore, in an actual fluorescence experiment, calculation is often performed taking into account the disturbance of the illumination light, or the calculated value of the shape of the spot illumination is often corrected based on a result of a preliminary experiment or the like.

  Therefore, in the fluorescence observation method using light stimulation or partial fading, the illumination area becomes large in both the thickness direction and the planar direction, and illumination light is irradiated to an unnecessary area of the specimen. Further, in the fluorescence observation method using FCS, if the size of the illumination area is different from the calculated value, there is a possibility that an erroneous analysis result is obtained.

  The present invention has been made in view of such a situation, and enables the spot illumination with a small illumination area and extremely thin in the optical axis direction to be performed, and the distribution shape of the illumination area is theoretically calculated. It is intended to be a sensible size.

  The illumination device for a microscope according to one aspect of the present invention includes a light source, a collimator lens that changes the illumination light emitted from the light source into a parallel light beam, and an objective lens, and performs illumination for total reflection on a specimen through the objective lens. In the apparatus, the illumination light converted into a parallel light beam by the collimator lens is incident on a predetermined range on the image side focal plane of the objective lens, and is condensed on the surface of the cover glass that protects the sample. Thus, it is configured so as to be totally reflected on the surface on the sample side.

  The light source may be a laser light source, and may be guided to the collimator lens using a single mode fiber, and the single mode fiber emitting portion or the collimator lens may be mutually in the optical axis direction and perpendicular to the optical axis. It can be configured to be adjustable in various directions and rotational directions.

  The exit portion of the single mode fiber, the collimator lens, and the diaphragm arranged on the sample side of the collimator lens can constitute a light source unit, and the entire light source unit is configured to be movable in a direction perpendicular to the optical axis. be able to.

  In one aspect of the present invention, the illumination light converted into a parallel light beam by the collimator lens is incident on a predetermined range on the image-side focal plane of the objective lens, and is condensed on the surface of the cover glass that protects the sample. By doing so, it is totally reflected on the surface on the sample side.

  According to the present invention, the illumination area is small because the illumination spot is formed by condensing the illumination light on the specimen surface side of the cover glass in the total reflection illumination that illuminates a shallow area in principle using evanescent light. Spot illumination with high accuracy can be performed with the distribution shape of the illumination area having a theoretical size.

  Embodiments of the present invention will be described below. Correspondences between the constituent elements of the present invention and the embodiments described in the specification or the drawings are exemplified as follows. This description is intended to confirm that the embodiments supporting the present invention are described in the specification or the drawings. Therefore, even if there is an embodiment which is described in the specification or the drawings but is not described here as an embodiment corresponding to the constituent elements of the present invention, that is not the case. It does not mean that the form does not correspond to the constituent requirements. Conversely, even if an embodiment is described here as corresponding to a configuration requirement, that means that the embodiment does not correspond to a configuration requirement other than the configuration requirement. It's not something to do.

  The microscope illumination apparatus according to one aspect of the present invention (for example, the illumination optical system of the microscope 1 in FIG. 1, the microscope 101 in FIG. 4, or the microscope 201 in FIG. 5) is a light source (for example, the laser light source 51 in FIG. The light source 54, the laser box 121 in FIG. 4 or the laser box 211 in FIG. 5 and a collimator lens (for example, the field lens 16 in FIG. 1, the field lens 16 in FIG. 4) that makes the illumination light emitted from the light source a parallel light beam. 5 or a relay lens 217) and an objective lens (for example, the objective lens 18 in FIG. 1, FIG. 4, or FIG. 5), and a microscope illumination device that performs total reflection illumination on a specimen through the objective lens. A cover that protects the specimen by causing the illumination light, which has been converted into a parallel light beam by the collimator lens, to enter a predetermined range on the image-side focal plane of the objective lens Las (e.g., FIG. 1, the cover glass 5 of FIG. 4 or FIG. 5) by condensing the surface of the specimen side, and configured so as to totally reflected by the surface of the specimen side.

  The light source is a laser light source, and is guided to the collimator lens using a single mode fiber (for example, the single mode fiber 12 in FIG. 1), and the single mode fiber emitting portion (for example, the emitting portion 12B in FIG. 1) or The collimator lenses are configured to be mutually adjustable in an optical axis direction, a direction perpendicular to the optical axis, and a rotation direction.

  The exit portion of the single mode fiber, the collimator lens, and a diaphragm (for example, the diaphragm 62 in FIG. 1) disposed on the sample side of the collimator lens constitute a light source unit (for example, the light source unit 13 in FIG. 1). The entire light source unit is configured to be movable in the direction perpendicular to the optical axis.

  Embodiments to which the present invention is applied will be described below with reference to the drawings.

  FIG. 1 is a diagram showing an embodiment of an illumination optical system of a microscope to which the present invention is applied. The illumination optical system of the microscope 1 in FIG. 1 includes a laser unit 11, a single mode fiber 12, a light source unit 13, a relay lens 14, a dichroic mirror 15, a field lens 16, a dichroic mirror 17, an objective lens 18, a single mode fiber 21, and a collimator. The lens 22, mercury lamp 31, collimator lens 32, electric shutter 33, relay lens 34, field stop 35, field lens 36, excitation filter 37, dichroic mirror 38, barrier filter 41, and second objective lens 42 are included. Composed.

  Among these, the laser illumination unit 11, the single mode fiber 12, the light source unit 13, the relay lens 14, the field lens 16, the dichroic mirror 17, and the objective lens 18 constitute a spot illumination optical system. Further, a TIRF illumination optical system is constituted by a laser light source (not shown), a single mode fiber 21, a collimator lens 22, a dichroic mirror 15, a field lens 16, a dichroic mirror 17, and an objective lens 18. Further, an epi-illumination optical system is configured by the mercury lamp 31, the collimator lens 32, the electric shutter 33, the relay lens 34, the field stop 35, the field lens 36, the excitation filter 37, the dichroic mirror 38, and the objective lens 18.

  In FIG. 1, the specimen 2 to be observed by the microscope 1 is immersed in the solution 4 in the slide glass 3 and protected by the cover glass 5. Further, oil 6 is filled in a portion of the objective lens 18 that contacts the cover glass 5.

  The laser unit 11 includes a laser light source 51 that emits a laser beam having a wavelength of 488 nm (hereinafter also referred to as a first spot laser beam), a shutter 52, a mirror 53, and a laser beam having a wavelength of 547 nm (hereinafter also referred to as a second spot laser beam). And a shutter 55, and a dichroic mirror 56 having a characteristic of transmitting light having a wavelength of 488 nm and reflecting a wavelength of 547 nm. Further, the laser unit 11 is connected to an incident portion 12A of a single mode fiber 12.

  Hereinafter, when it is not necessary to distinguish between the first spot laser beam and the second spot laser beam, they are simply referred to as spot laser beams.

  The incident portion 12A of the single mode fiber 12, the laser light source 51, the mirror 53, the laser light source 54, and the dichroic mirror 56 are provided with an adjustment mechanism (not shown). By adjusting each adjustment mechanism, the first spot laser light emitted from the laser light source 51 is reflected by the mirror 53, passes through the dichroic mirror 56, and is incident on the incident portion 12A of the single mode fiber 12 as one light beam. Led. Further, by adjusting each adjustment mechanism, the second spot laser light emitted from the laser light source 54 is reflected by the dichroic mirror 56 and guided to the incident portion 12A of the single mode fiber 12 as one light beam. Further, by controlling the opening and closing of the shutter 52 and the shutter 55 from the outside, on / off of the first spot laser beam and the second spot laser beam is controlled.

  In the laser unit 11 of FIG. 1, two laser light sources are provided. However, three or more laser light sources may be provided. In this case, for example, it can be realized by newly adding a combination of a laser light source, a shutter, a dichroic mirror, and an adjustment mechanism.

  Further, in FIG. 1, the single mode fiber 21 and the collimator lens 22 of the TIRF illumination optical system are configured like the single mode fiber 12 to the relay lens 14 of the spot illumination optical system, thereby realizing two-point thin spot illumination. it can. Similarly, this illumination optical system can be increased together with the dichroic mirror.

  The incident part 12A of the single mode fiber 12 is connected to the laser unit 11 as described above, and the emitting part 12B is connected to the light source unit 13. The single mode fiber 12 emits the spot laser light incident from the incident portion 12 </ b> A from the emitting portion 12 </ b> B provided in the light source unit 13.

  The light source unit 13 is configured to include a collimator lens 61 and a diaphragm 62. The spot laser light emitted from the emitting portion 12B of the single mode fiber 12 is converted into a parallel light beam by the collimator lens 61, passes through the diaphragm 62, and enters the relay lens 14. The diaphragm diameter of the diaphragm 62 may be either fixed or variable.

  In addition, the emission part 12B of the single mode fiber 12 can be moved in an optical axis direction and a direction perpendicular to the optical axis by an adjusting mechanism (not shown), and the position can be adjusted. That is, it is possible to adjust the distance between the emitting portion 12B and the collimator lens 61 and the position of the spot laser light incident on the collimator lens 61. Further, the emission unit 12B can be rotated around the exit of the spot laser beam by an angle adjustment mechanism (not shown), and the angle at which the spot laser beam is emitted can be adjusted.

  Further, the light source unit 13 itself can also be moved in a direction perpendicular to the optical axis by an adjustment mechanism (not shown), and the position of the spot laser light incident on the relay lens 14 can be adjusted.

  It should be noted that some or all of the functions of the adjusting mechanism and the angle adjusting mechanism of the emitting portion 12B can be provided on the collimator lens 61 side. That is, the collimator lens 61 can be moved or rotated in the direction perpendicular to the optical axis and the optical axis.

  The spot laser light emitted from the light source unit 13 is once condensed by the relay lens 14, passes through the dichroic mirror 15, and becomes a parallel light beam again by the field lens 16. Further, the spot laser light converted into a parallel light beam is reflected by the dichroic mirror 17 in the optical axis direction of the objective lens 18 and enters the image-side focal plane (pupil plane) BF of the objective lens 18. Then, the spot laser light is condensed on the surface of the sample 2 that contacts the cover glass 5 by the objective lens 18, and the sample 2 is spot-illuminated. In this manner, total reflection spot illumination with a spot laser beam is realized. Details of the operation of the spot illumination optical system will be described later with reference to FIGS.

  Laser light emitted from a laser light source (not shown) of the TIRF illumination optical system (hereinafter also referred to as TIRF laser light) is incident on the single mode fiber 21 and is emitted from the emitting portion 21A in the optical axis direction of the collimator lens 22. . The TIRF laser light emitted from the single mode fiber 21 is converted into a parallel light beam by the collimator lens 22, reflected by the dichroic mirror 15 in the optical axis direction of the field lens 16, and transmitted through the field lens 16. At this time, the field lens 16 acts as a condensing lens, and the TIRF laser light transmitted through the field lens 16 is reflected by the dichroic mirror 17 in the optical axis direction of the objective lens 18 and the image side focal plane (pupil) of the objective lens 18 is reflected. Surface) Condensation in a predetermined region of BF. Then, the TIRF laser beam condensed in a predetermined region on the image side focal plane (pupil plane) BF of the objective lens 18 becomes a parallel light beam by the objective lens 18 and irradiates the sample 2. In this way, total reflection illumination by TIRF laser light is realized.

  Note that the emission unit 21A of the single mode fiber 21 is also provided with an adjustment mechanism and an angle adjustment mechanism (not shown) similar to those of the emission unit 12B of the single mode fiber 12, and moves in the optical axis direction and the direction perpendicular to the optical axis. , And rotation around the injection port of the injection portion 21A is possible.

  Light emitted from the mercury lamp 31 of the epi-illumination optical system (hereinafter also referred to as epi-illumination light) is converted into a parallel light beam by the collimator lens 32 and passes through the electric shutter 33, the relay lens 34, the field stop 35, and the field lens 36. The excitation filter 37 selectively transmits only a predetermined wavelength component. The incident illumination light that has passed through the excitation filter 37 is reflected by the dichroic mirror 38 in the optical axis direction of the objective lens 18, passes through the dichroic mirror 17 and the objective lens 18, and is irradiated onto the sample 2.

  The electric shutter 33 can be opened and closed from the outside, and the illumination of the epi-illumination optical system can be switched on or off.

  The fluorescence emitted from the specimen 2 or the reflected light reflected by the specimen 2 by irradiating the first spot laser light, the second spot laser light, the TIRF laser light, or the epi-illumination light is collected by the objective lens 18 and is dichroic. By the mirror 17 and the dichroic mirror 38, only components necessary for observation are transmitted, and unnecessary components are reflected. The light having a predetermined wavelength among the light transmitted through the dichroic mirror 38 passes through the barrier filter 41 and forms an image on the image plane IM by the second objective lens 42.

  Next, the details of the operation of the spot illumination optical system will be described with reference to FIGS.

  2 and 3 are diagrams in which portions related to the spot illumination optical system are extracted from the illumination optical system of the microscope 1 in FIG. However, illustration of the laser unit 11 is omitted in FIGS. 2 and 3.

  In the following, the core diameter of the exit port of the exit portion 12B of the single mode fiber 12 is set to 3.5 μm, and the single mode fiber 12 is spot laser light from the exit portion 12B under the condition of NA = 0.11 (= about 13 degrees angle). Is assumed to be emitted.

  FIG. 2 shows an example in which adjustment is made so that the emission center of the fiber exit end face of the spot laser light is positioned on the optical axis of the optical system.

  The brightness of the spot laser light emitted from the emitting portion 12B at an angle of about 13 degrees is not uniform, and becomes brighter in the center and darker in the periphery according to a Gaussian distribution. For this reason, the diaphragm 62 is used when dark ambient light is limited. For example, the radiation range of the spot laser light is limited to about a half angle by the diaphragm 62.

  The spot laser beam that has passed through the diaphragm 62 is once condensed by the relay lens 14 and further converted into a parallel beam by the field lens 16. The spot laser beam converted into a parallel beam by the field lens 16 is reflected by the dichroic mirror 17 in the optical axis direction of the objective lens 18 and becomes a parallel beam on the image-side focal plane (pupil plane) BF of the objective lens 18. Spot laser light arrives. Thereby, the spot laser beam is condensed on the focal plane of the objective lens 18 on the sample 2 side.

  FIG. 3 shows an example in which the light source unit 13 is translated by a distance d31 in a direction indicated by an arrow A31 perpendicular to the optical axis of the laser light, as compared with FIG.

By moving the light source unit 13 in parallel, the path of the spot laser beam is shifted to the right in the drawing from the center of the optical axis of the objective lens 18, and the spot laser beam is reflected on the image side focal plane (pupil plane) of the objective lens 18. ) Pass the end of BF. When the position where the spot laser beam passes through the image side focal plane (pupil plane) BF of the objective lens 18 is NA = 1.38 or more, the spot laser beam is condensed on the surface of the cover glass 5 on the sample 2 side. together is totally reflected in a state in which total internal reflection of the spot illumination is performed on the sample 2 (however, the refractive index of the cover glass 5 n _cov = 1.5255, refractive index n _obj = 1.33 from the cover glass 5 specimens side of the medium And).

Here, a specific example will be given to consider the conditions under which the spot laser light is totally reflected and the irradiation range of the spot illumination by the spot laser light. Hereinafter, the core diameter φs = 3.5 μm, the numerical aperture NAs = 0.11, the focal length fc = 5 mm of the collimator lens 61, the focal length fr = 1000 mm of the relay lens 14, and the field lens 16 The focal length f _f = 200 mm, the magnification M _obj of the objective lens 18 = 100, the numerical aperture NA _obj = 1.49, and the focal length f _obj = 2 mm.

In this case, the objective lens satisfying the condition of total reflection in the range from NA = 1.38 to NA = 1.49 on the image side focal plane (pupil plane) BF of the objective lens 18, that is, the surface of the cover glass 5 on the specimen 2 side. Total reflection illumination is realized when the spot laser beam passes through a range on the 18 image-side focal plane (pupil plane) BF. That is, the spot laser beam has a width (TIRF of 0.22 mm (= f _obj × (1.49−1.38) = 2 × (1.49−1.38)) at the edge of the image side focal plane (pupil plane) BF of the objective lens 18 (TIRF). Total reflection illumination is realized when it passes through (range).

On the other hand, the size (diameter) of the light beam of the spot laser light emitted from the single mode fiber 12 is φ1.1 mm (= fc × NAs × 2 = 5 × 0.11 × 2) when it exits the collimator lens 61. . Furthermore, the size of the light beam spot laser beam is transmitted through the relay lens 14 and the field lens 16, is reduced to _{φ0.22mm (= φ1.1mm × f f /fr=φ1.1mm×200/1000} ) The That is, the size of the light beam of the spot laser light falls within the width of the TIRF range.

The NA _SL of the spot laser light that irradiates the specimen 2 from the objective lens 18 is obtained by the following equation (1) from the focal length of the single mode fiber 12 and each lens.

NA _SL = NAs × fc / fr × f _f / f _obj = 0.11 × 5/1000 × 200/2 = 0.055 (1)

The irradiation range of the spot laser beam when the sample 2 is irradiated is obtained by the following equation (2) using this NA _SL .

Irradiation range = (n x representative wavelength of spot laser beam) / (2 x NA _SL )
= (1.55 x representative wavelength of spot laser beam) / (2 x 0.055) (2)
Where n: representative value of refractive index of oil 6 and cover glass 5

  Accordingly, the irradiation ranges of the first spot laser beam and the second spot laser beam are about 7 μm and about 8 μm, respectively, from the following expressions (3) and (4).

First spot laser beam irradiation range = (1.55 x 488nm) / (2 x 0.055)
≒ 6.9μm ≒ approx. 7μm (3)
Second spot laser beam irradiation range = (1.55 x 547 nm) / (2 x 0.055)
≒ 7.7μm ≒ Approximately 8μm (4)

  In addition, it is known that the thickness of the total reflection illumination through the objective lens 18 by the first spot laser beam or the second spot laser beam is about 0.15 μm (the thickness of the evanescent light in which the evanescent light works as the illumination light). .

  It is possible to adjust the irradiation range of the spot illumination by adjusting the size of the light beam of the spot laser light emitted from the single mode fiber 12.

  As described above, spot illumination can be performed in a range called an evanescent layer on the surface of the specimen 2 in contact with the cover glass 5. Further, in theory of total reflection illumination, the spot illumination area and thickness are close to theoretical values regardless of the refractive index of the sample 2 and the light scattering state, so that highly accurate spot illumination can be performed.

  As a result, in the fluorescence observation method using light stimulation or FRAP, the range in which the specimen 2 is irradiated with the illumination light can be narrowed, and the specimen 2 can be efficiently illuminated and the damage to the specimen 2 caused by the illumination can be reduced. Can do.

  Here, consider the case of performing fluorescence observation by FCS using the microscope 1.

As described above, in the fluorescence observation method using FCS, the volume of the illumination light irradiation range needs to be several fl (ferm · liter) or less. For example, in the case of a condensing size with a normal objective lens, in the case of a water immersion type objective lens with a magnification of 100 times and NA = 1.2, the illumination width in the direction perpendicular to the optical axis is about 0.3 μm, The illumination thickness is calculated to be about 2 μm. When the condensing portion is calculated as a cylinder, the volume is about 0.14 fl (= 0.15 ² × π × 2 × 10 ⁻⁹ (mm ² )).

On the other hand, when the second spot laser beam is used in the microscope 1, the diameter of the irradiation area of the spot illumination is about 7.7 μm and the thickness is 0.15 μm, so the volume is about 7 fl (= 3.85 ² × π × 0.15 × 10 ^-9 (mm ² )). Compared to the previous example, the volume of the irradiation range is large, but it is a few fl level. Therefore, it is sufficiently possible to perform fluorescence observation with the microscope 1 using FCS.

  FIG. 4 shows an embodiment of the illumination optical system of the microscope 101 in which the light source (laser unit 11, single mode fiber 12, and light source unit 13) of the spot illumination optical system of the microscope 1 of FIG. Is shown. In the figure, portions corresponding to those in FIG. 1 are denoted by the same reference numerals, and description of portions having the same processing will be omitted because it will be repeated.

  The light source unit 111 is configured to include a laser box 121, a beam expander 122, and a diaphragm 123. Laser light emitted from the laser box 121 (hereinafter referred to as spot laser light) is incident on the beam expander 122, the diameter of which is enlarged, and a light beam having a predetermined size is formed by the diaphragm 123 and incident on the relay lens 14. To do. The operation after the relay lens 14 is the same as that of the microscope 1.

  The laser box 121 can be rotated around the emission port by an angle adjustment mechanism (not shown) and adjust the angle at which the spot laser beam is emitted, similarly to the emission part 12B of the single mode fiber 12 of the microscope 1. It is possible.

  Similarly to the light source unit 13 of the microscope 1, the light source unit 111 itself can be moved perpendicular to the optical axis by an adjustment mechanism (not shown), and adjusts the position of the spot laser light incident on the relay lens 14. It is possible.

  FIG. 5 shows an embodiment of the illumination optical system of the microscope 201 that can form a plurality of spots on the specimen surface using an optical element that changes the angle of the spot laser beam and a computer-controlled diffraction diffusion plate. In the figure, portions corresponding to those in FIG. 1 are denoted by the same reference numerals, and description of portions having the same processing will be omitted because it will be repeated.

  5 includes a laser box 211, a shutter 212, a steerer control unit 213, a computer controlled diffraction generator 214, a diaphragm 215, a relay lens 216, a relay lens 217, a dichroic mirror 17, and an objective lens. 18, a barrier filter 41, and a second objective lens 42. The steerer control unit 213 is configured to include a first steerer 231 and a second steerer 232. The first steerer 231 and the second steerer 232 each independently adjust the angle with respect to a laser beam emitted from the laser box 211 (hereinafter referred to as a spot laser beam) and a spot laser beam by control from the outside. It is possible to adjust the position in the direction perpendicular to the optical axis.

  The spot laser light emitted from the laser box 211 passes through the shutter 212, is reflected by the first steerer 231 and the second steerer 232, and enters the diffraction generating element 214. As described above, the first steerer 231 and the second steerer 232 can adjust the angle and position with respect to the spot laser beam, and the spot laser incident on the diffraction generating element 214 can be adjusted by adjusting each angle and position. It is possible to freely change the direction and position of light.

  In FIG. 5, the laser box 211, the shutter 212, the first steerer 231, and the second steerer 232 are drawn on the same plane, but depending on the emission direction of the spot laser light from the laser box 211, respectively. The positional relationship of changes. For example, the laser box 211 emits spot laser light in a direction perpendicular to the paper surface (a direction penetrating from the top to the bottom of the paper surface), and the first steerer 231 emits the spot laser light toward the paper surface of the second steerer 232. It is conceivable that the second steerer 232 reflects the spot laser light in the direction of the relay lens 216 in the drawing.

  Here, with reference to FIG. 6, the spot formation by the diffraction generating element 214 will be described. FIG. 6 shows only the portion related to the description extracted from the illumination optical system of the microscope 201 shown in FIG. For convenience of explanation, FIG. 6 shows that the diffraction generating element 214, the relay lens 216, the relay lens 217, the dichroic mirror 17, and the objective lens 18 are on the same optical axis. Each component is arranged as shown in FIG.

  When the diffraction generating element 214 forms a spot Sa1 at the top of the circumference C1 of the field surface 214A in the drawing, the spot laser light having the spot Sa1 as a secondary light source passes through the center of the optical axis of the relay lens 216. The traveling direction changes in the optical axis direction of the relay lens 217 by the relay lens 217. The spot laser light is reflected by the dichroic mirror 17 in the direction of the optical axis of the objective lens 18 to form a spot Sa2 at the lowermost portion of the circumference C2 of the image side focal plane (pupil plane) BF of the objective lens 18 in the drawing. To do. Similarly, when the diffraction generating element 214 forms a spot Sb1 in the lowermost portion of the view of the circumference C1 on the field surface 214A, the view of the circumference C2 of the image side focal plane (pupil plane) BF of the objective lens 18 is shown. A spot Sb2 is formed on the uppermost part.

  Accordingly, the position of the circumference C1 is set so that the circumference C2 falls within the TIRF range of the image side focal plane (pupil plane) BF of the objective lens 18, and the angles and positions of the first steerer 231 and the second steerer 232 are set. And a spot is formed by the diffraction generating element 214 at an arbitrary position on the circumference C1 to realize total reflection spot illumination. Then, the angle and position of the first steerer and the second steerer are adjusted, and the incident angle of the spot laser beam incident on the spot is adjusted without changing the position of the spot on the circumference C1, whereby total reflection illumination is achieved. The position of the spot illumination on the specimen 2 can be moved while maintaining this state.

  Moreover, it becomes possible to form illumination of an arbitrary shape by continuously moving the spot at a high speed. Further, by linking with the switching of the shutter 212 on or off, spot illumination of a plurality of points can be realized. For example, in fluorescence observation using FCS, observation of a plurality of points of a specimen becomes possible.

  In addition, by adjusting the angle and position of the spot laser beam with a high-speed element such as a galvanometer mirror or AOM (Acousto-Optic Modulator), not only the spot movement can be made faster, but also the spot at multiple points. Lighting can be performed almost simultaneously.

  Furthermore, the confocal of total reflection illumination is realizable by scanning a spot illumination part. In addition, it is possible to reliably realize illumination with a thinner thickness in the optical axis direction (thickness direction) than conventional confocals. For example, conventionally, when an objective lens having a magnification of 100 and an NA of 1.4 is used, the illumination thickness is about 0.4 μm, but in the present invention, the thickness can be about 0.15 μm.

  In the above description, an example in which laser light is used has been described. However, in the embodiment of the present invention, for example, light of another type of light source such as a xenon lamp or a mercury lamp is used to increase the size of a light beam by a diaphragm or the like. You may make it use it, restrict | squeezing so that may become small enough.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

It is a figure which shows 1st Embodiment of the illumination optical system of the microscope to which this invention is applied. It is a figure for demonstrating the detail of operation | movement of a spot illumination optical system. It is a figure for demonstrating the detail of operation | movement of a spot illumination optical system. It is a figure which shows 2nd Embodiment of the illumination optical system of the microscope to which this invention is applied. It is a figure which shows 3rd Embodiment of the illumination optical system of the microscope to which this invention is applied. It is a figure for demonstrating adjustment of the position of spot illumination.

Explanation of symbols

  1 microscope, 2 specimens, 3 slide glass, 5 cover glass, 6 oil, 11 laser unit, 12 single mode fiber, 13 light source unit, 14 relay lens, 16 field lens, 17 dichroic mirror, 18 objective lens, 51 laser light source, 54 laser light source, 55 shutter, 61 collimator lens, 62 aperture, 101 microscope, 111 light source unit, 121 laser box, 122 beam expander, 123 aperture, 201 microscope, 211 laser box, 212 shutter, 213 steerer control unit, 214 diffraction Generating element, 215 stop, 216, 217 relay lens

Claims (3)

In a microscope illumination apparatus, comprising: a light source; a collimator lens that converts illumination light emitted from the light source into a parallel luminous flux; and an objective lens, and performing total reflection illumination on a specimen through the objective lens.
By making the illumination light converted into a parallel light beam by the collimator lens into a predetermined range on the image side focal plane of the objective lens, and condensing on the surface on the sample side of the cover glass protecting the sample, An illumination device for a microscope configured to totally reflect on the surface on the specimen side. The light source is a laser light source, and is guided to the collimator lens using a single mode fiber, and the exit part of the single mode fiber or the collimator lens is mutually in the optical axis direction, the direction perpendicular to the optical axis, and the rotation direction. The illumination device for a microscope according to claim 1, wherein the illumination device is adjustable. The single-mode fiber emitting portion, the collimator lens, and a diaphragm disposed on the specimen side of the collimator lens constitute a light source unit, and the entire light source unit is configured to be movable in a direction perpendicular to the optical axis. Item 3. The illumination device for a microscope according to Item 2.

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