JP2010060854A - Microscopic device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscopic device, capable of correcting a change in polarization state caused by an optical system constituting the microscopic device. <P>SOLUTION: The microscopic device 100 includes: a light source 1 which emits illuminating light L1; an objective lens 4 which gives the illuminating light L1 transmitted through an area with NA of 1 or more to a sample 30; a polarizer 5 which converts the illuminating light L1 to a linearly polarized state; a condenser lens 6 which collects the illuminating light L1 transmitted by the polarizer 5 to a pupil surface 4a of the objective lens 4; an imaging lens 8 which collects observation light L2 emitted from the sample 30 and transmitted by the objective lens 4 to form an image of the sample 30; an analyzer 9 disposed so that its polarization surface for the transmitted light is orthogonal to the polarizer 5; and a first compensator 7 disposed between the polarizer 5 and the condenser lens 6. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a microscope apparatus.

Conventionally, in biology research, illumination using total reflection (TIRF illumination) is often used for the excitation method of fluorescent dyes, and an invention relating to a total reflection illumination fluorescence microscope apparatus (TIRF microscope) using this has been proposed. (For example, refer to Patent Document 1). There is also known a polarization microscope apparatus used for observing a sample having birefringence using polarized light. Such a polarizing microscope apparatus is configured by arranging two polarizers (polarizers) on the illumination side and the imaging side with their transmission axes (optical axes) orthogonal to each other. ), An object that changes the polarization state between these two polarizers (polarizers), that is, a sample having birefringence, and the polarization state is changed to change the polarization state of the imaging side (polarizer). (This is referred to as an “analyzer”) and the characteristics of the sample are observed by observing light that has passed through.
Japanese Patent Laid-Open No. 2004-85796

  However, in the conventional methods including Patent Document 1, a method for maintaining the polarization state of illumination light or observation light in TIRF illumination or epi-illumination is not clearly described. Therefore, when polarized light passes through optical elements such as a lens of an illumination optical system and an objective lens, a change in polarization state such as a phase change occurs due to the influence of these lenses. As a result, there has been a problem that linearly polarized light becomes elliptically polarized and the contrast of the image is lowered. This change in the polarization state is particularly remarkable due to the influence of the coating (antireflection film or the like) applied to the lens.

  The present invention has been made in view of the above problems, and illumination light by an optical element constituting an optical system such as a condensing lens and an objective lens by disposing a compensator between the condensing lens and the polarizer. An object of the present invention is to provide a microscope apparatus capable of realizing illumination with a good polarization state by correcting the elliptical polarization of the light and maintaining the linear polarization state of the illumination light transmitted through the condenser lens and the objective lens. And

  In order to solve the above problems, a microscope apparatus according to the present invention includes a light source that emits illumination light, an objective lens that irradiates a sample with illumination light that passes through a position greater than a predetermined NA, and a light source and an objective lens. A polarizer that is arranged in between and converts the illumination light into a linearly polarized state, a condenser lens that condenses the illumination light transmitted through the polarizer on the pupil plane of the objective lens, and an observation light that exits the sample and passes through the objective lens An imaging lens that collects an image of the sample to form an image of the sample, an analyzer that is disposed between the imaging lens and the image and that has a plane of polarization of transmitted light orthogonal to the polarizer, A first compensator which is arranged between the lens and the polarizer and which corrects the elliptical polarization of the illumination light by the optical element through which the illumination light passes, and maintains the linear polarization state of the illumination light applied to the sample Is done.

  In the microscope apparatus according to the present invention, it is preferable that the illumination light passes through a region having an NA of 1 or more in the objective lens.

  The microscope apparatus according to the present invention includes an illumination light detection unit that detects a polarization state of illumination light transmitted through the objective lens, and elliptically polarized light that calculates the degree of elliptical polarization of the illumination light detected by the illumination light detection unit. It is preferable that a phase difference is given to the illumination light by the first compensator so as to cancel the deviation amount of the elliptical polarization of the illumination light calculated by the elliptical polarization state calculation unit.

  At this time, in the microscope apparatus according to the present invention, it is preferable that the first compensator is composed of a Babinet-Soleil compensator.

  Alternatively, in the microscope apparatus according to the present invention, it is preferable that the first compensator is composed of a Brace-Kohler compensator.

  The microscope apparatus according to the present invention preferably includes a second compensator that is disposed between the imaging lens and the analyzer and corrects the elliptical polarization of the observation light by the objective lens.

  In the microscope apparatus according to the present invention, it is preferable that the correction amount of the second compensator is the same as that of the first compensator.

  At this time, in the microscope apparatus according to the present invention, it is preferable that the second compensator is constituted by a Babinet-Soleil compensator.

  Alternatively, in the microscope apparatus according to the present invention, it is preferable that the second compensator is composed of a Brace-Kohler compensator.

  When the microscope apparatus according to the present invention is configured as described above, when illumination is performed with declinated falling illumination and polarized light, illumination by an optical element through which illumination light passes by the first compensator provided in the illumination optical system By correcting the elliptical polarization of light and maintaining the linear polarization state of the illumination light, illumination with a good polarization state can be realized, and fluorescence polarization observation can be performed with high contrast.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows an arrangement of optical systems in the microscope apparatus 100 of the present embodiment. The microscope apparatus 100 includes an illumination optical system 2 that illuminates a sample 30 with illumination light (laser light) L1 emitted from a light source 1 via an objective lens 4, and an observation lens L2 emitted from the sample 30 as an objective lens. 4 and an observation optical system 3 which focuses and forms an image, and observes an image of the sample 30 by fluorescence (observation light L2) radiated from the sample 30 using total reflection illumination. is there.

  The illumination optical system 2 constituting the microscope apparatus 100 is disposed between the objective lens 4 that irradiates the sample 30 with illumination light L1 that is a substantially parallel light beam emitted from the light source 1, and between the light source 1 and the objective lens 4. The polarizer 5 that converts the illumination light L1 into a linearly polarized state, the condenser lens 6 that condenses the illumination light L1 that has passed through the polarizer 5 on the pupil plane 4a of the objective lens 4, the polarizer 5 and the condenser lens 6 And a first compensator 7 disposed between the two. On the other hand, the observation optical system 3 condenses the observation light L2 emitted from the sample 30 to be a substantially parallel light beam, and condenses the observation light L2 transmitted through the objective lens 4 to collect an image of the sample 30. An imaging lens 8 that forms an image of I, an analyzer 9 that is disposed between the imaging lens 8 and the image I, and has a polarization plane of transmitted light orthogonal to the polarizer 5, and the imaging lens 8 And a second compensator 10 disposed between the analyzer 9 and the analyzer 9.

  A sample 30 to be observed is accommodated together with a culture solution 32 in a petri dish 31 placed on a stage (not shown). A cover glass 33 is attached to the upper surface of the sample 30, and a liquid 34 for immersion is filled between the cover glass 33 and the objective lens 4.

  As shown in FIG. 2, in this microscope apparatus 100, the illumination light (laser light) L1 emitted from the light source 1 has an outer peripheral portion of the objective lens 4 as a light beam having a small diameter, specifically, a numerical aperture (NA). The sample 30 is configured to be incidentally illuminated through one or more regions. At this time, the angle formed by the optical axis when the illumination light L1 is irradiated onto the sample 30 is set to an angle at which total reflection occurs at the boundary between the cover glass 33 and the sample 30. However, the illumination light L1 is not completely reflected at this boundary, but a very small amount of light oozes out from the cover glass 33 toward the sample 30 side. The light that exudes to the boundary side is evanescent light, and the amount of exudation in the depth direction of the sample 30 is about the wavelength of the light source to be used. Therefore, when evanescent light is used as illumination light, the illumination range is limited to the depth of the wavelength of the light source to be used, so the portion that emits fluorescence (observation light L2) is extremely narrow and otherwise does not emit fluorescence. Unlike normal epi-illumination, the background fluorescence is very small. Therefore, extremely high S / N ratio can be realized by total reflection illumination, and it is particularly effective for observation of the cell membrane surface and visualization of a single fluorescent dye molecule located near the cover glass surface.

  In the illumination optical system 2 of the microscope apparatus 100, the shutter 11 that controls transmission and blocking of the illumination light L1 from the light source 1 and the illumination light L1 that is a substantially parallel light beam are expanded to a parallel light beam having a constant diameter. An expander lens 12, an ND filter 13 for adjusting the amount of the illumination light L1, a wave plate (1/2 wavelength) 14 for changing the direction of the linearly polarized illumination light L1 transmitted through the polarizer 5, and light Herving glasses 15 and 16 that perform parallel movement of the axes, and a first mirror 17 that reflects the illumination light L1 collected by the condenser lens 6 in the direction of the objective lens 4 are provided. In addition, the observation optical system 3 includes a stop 18 that allows the observation light L2 that is a substantially parallel light beam that has passed through the objective lens 4 to pass therethrough, and a first light that reflects the observation light L2 that has passed through the stop 18 in the direction of the imaging lens 8. 2 mirrors 19 are provided. Here, the herving glasses 15 and 16 and the first mirror 17 are configured to be movable and fixed in a direction parallel to the optical axis by an appropriate driving mechanism (not shown).

  In such a microscope apparatus 100, as shown in FIG. 1, the illumination light L1, which is a substantially parallel light beam emitted from the light source 1, passes through the shutter 11, and then is expanded to a parallel light beam having a predetermined diameter by the expander lens 12. Then, it passes through the ND filter 13 and is converted into a linearly polarized state by the polarizer 5. The linearly polarized illumination light L1 transmitted through the polarizer 5 is changed in direction by the wave plate 14, then transmitted through the first compensator 7, collected by the condensing lens 6, and the herving glasses 15, 16 After the position from the optical axis is adjusted, the light is reflected by the first mirror 17 and travels toward the objective lens 4. In this way, the illumination light L1 is condensed on the pupil plane 4a of the objective lens 4 by the condenser lens 6, and illuminates the sample 30 through the objective lens 4 as described above.

  The fluorescent material in the sample 30 emits fluorescence (observation light) by the illumination light L1 irradiated to the sample 30 through the objective lens 4. The observation light L2 is condensed by the imaging lens 8 and is reflected on the sample 30. An image I is formed and observed. In this case, the observation light L 2 emitted from the sample 30 passes through the objective lens 4, passes through the diaphragm 18, is reflected by the second mirror 19, and is collected by the imaging lens 8. The collected observation light L2 further passes through the second compensator 10 and the analyzer 9, and forms an image I on the imaging surface.

  In such a microscope apparatus 100, the polarizer 5 and the analyzer 9 are arranged so that their transmission axes are orthogonal (the above-mentioned “Cross Nicol”), and the polarization state is between the polarizer 5 and the analyzer 9. Without an object that changes (polarization plane tilt and retardation), linearly polarized light that has passed through the polarizer 5 cannot pass through the analyzer 9. On the other hand, since the fluorescence (observation light L2) emitted from the sample 30 has a different polarization state, it passes through the analyzer 9 and the observer can observe the image I.

  However, as described above, the polarized light passes through the condenser lens 6 and the objective lens 4 disposed between the polarizer 5 and the analyzer 9, so that the illumination light L1 and the observation light L2 are polarized by the influence of these lenses. The state changes, and the contrast of the image is lowered due to leakage of the illumination light L1 from the analyzer 9 or the like. In particular, in the high-magnification objective lens 4, as shown in FIG. 2A, the lens surface close to the sample 30 includes a surface with a strong curvature, and an antireflection film applied to such a lens surface. In many cases, a predetermined film thickness cannot be secured at a position away from the optical axis (a position where a light beam having an NA of 1 or more passes). For this reason, a light beam that passes through a region having an NA of 1 or more has a significant degree of elliptical polarization, and thus needs to be corrected. For this reason, in the microscope apparatus 100 according to the present embodiment, the first and second compensators 7 and 10 are used to correct the change in the polarization state generated in the condenser lens 9 and the objective lens 13. It is configured. A method for adjusting the positions of the first and second compensators 7 and 10 for this correction will be described below.

  When the illumination light beam reaches the entire optical path as in normal epi-illumination, it passes through the entire lens constituting the objective lens 4. In that case, different phase changes occur at different positions of the lens, and these are combined. In this state, it is difficult to perform phase compensation with a single compensator. On the other hand, in the microscope apparatus 100 of the present embodiment, as shown in FIG. 2, the size of the illumination light beam is limited, and a part of the outer peripheral portion of the objective lens 4 (specifically, in FIG. Since the NA is indicated by the oblique lines (the area where NA is 1 or more), the influence of the objective lens 4 and the like is almost the same amount, and it is possible to compensate with one compensator. That is, the polarization state is good by correcting the elliptical polarization of the illumination light L1 and the observation light L2 by the first and second compensators 7 and 10 arranged one by one in the illumination optical system 2 and the observation optical system 3, respectively. It realizes illumination and observation.

  The first compensator 7 disposed in the illumination optical system 2 corrects the elliptical polarization of the illumination light L1 by the objective lens 4 or the like so as to cancel the amount of elliptical polarization by the objective lens 4 or the like. The illumination light L1 is elliptically polarized so as to achieve a degree of conversion, whereby the linearly polarized state of the illumination light L1 transmitted through the objective lens 4 is maintained. The second compensator 10 arranged in the observation optical system 3 corrects the observation light L2 that has been elliptically polarized by the objective lens 4 to the original linearly polarized state. The elliptical polarization of the illumination light L1 by the optical system 100 is about 1/10 wavelength as a phase amount. Therefore, as the first and second compensators 7 and 10, a Babinet-Soleil compensator, A Brace-Kohler compensator may be used.

Here, the principle of the Babinet-Soleil compensator and the Brace-Kohler compensator will be described with reference to FIG. As shown in FIG. 3A, the Babinet-Soleil compensator is composed of a right crystal L and left crystals R1 and R2 divided into two prisms. Here, the right quartz crystal L and the left quartz crystals R1 and R2 are arranged in such a manner that two quartz plates L and R having the same thickness and polished in parallel to the optical axis are arranged in a direction in which the optical axes are orthogonal to each other. The structure is divided into prisms R1 and R2. Then, by moving one of the left quartz crystals R1, R2 in the optical axis direction, the amount of elliptical polarization of the light passing through the compensator is adjusted. On the other hand, the Brace-Kohler compensator measures a very small phase difference, and a mica plate having a phase difference ε = (1/20 to 1/60) × (2π / λ ₀ ) as the wave plate C. Is used. As shown in FIG. 3B, this Brace-Kohler compensator inserts a wave plate C between polarizing plates P and A arranged so that their optical axes are orthogonal, and rotates the wave plate C. Thus, the phase difference of the unknown linear birefringent material M is measured. By applying this principle, the amount of elliptically polarized light passing therethrough can be adjusted.

  In the microscope apparatus 100 according to the present embodiment, the first and second compensators 7 and 10 are configured to be rotatable around the optical axis by appropriate driving units 51 and 52 such as actuators, respectively. . In the control unit 50 that controls the operation of the driving units 51 and 52, the driving units 51 and 52 are driven in accordance with the state of elliptical polarization of the illumination light L1 and the observation light L2 in a direction to cancel the elliptical polarization. The first and second compensators 7 and 10 are rotated to perform correction.

  Such a microscope apparatus 100 according to the present embodiment further includes an illumination light detection unit 40 that detects the illumination light L1 transmitted through the objective lens 4. Between the illumination light detection unit 40 and the objective lens 4, a polarizing plate 41 that is rotatably controlled by the driving unit 53 is disposed. The control of the operation of the drive unit 53 is performed by the control unit 50. The control unit 50 calculates the amount of elliptical polarization of the illumination light L1 detected by the illumination light detection unit 40. It also has a function as Then, the control unit 50 operates the driving unit 53 to rotate the polarizing plate 41 to calculate the amount of elliptical polarization of the illumination light L1, and cancel the calculated amount of elliptical polarization of the illumination light L1. The driving units 51 and 52 are operated to adjust the positions of the first and second compensators 7 and 10. Below, the procedure of adjustment by the control part (elliptical polarization state calculation part) 50 is demonstrated.

  First, the irradiation direction of the illumination light L1 is changed by moving the first mirror 17 in the optical axis direction so that the illumination light detection unit 40 can detect the illumination light L1 (the illumination in this state). The light is called transmitted light L3). The transmitted light L3 transmitted through the polarizing plate 41 is received by the illumination light detection unit 40, and the signal strengths of the major axis and the minor axis of the transmitted light L3 are measured. The controller 50 calculates the amount of elliptical polarization based on the signal intensity information, and drives the drive unit 53 to rotate the polarizing plate 41 by a small amount in the direction to cancel the amount of elliptical polarization. Further, the signal intensity after rotation is measured by the illumination light detection unit 40, the control unit 50 calculates the degree of elliptical polarization again, and the polarizing plate 41 is rotated according to the degree of elliptical polarization.

  In this way, the signal intensity is measured by the illumination light detection unit 40, and the polarizing plate 41 is repeatedly rotated according to the degree of elliptical polarization calculated by the control unit 50, and finally the degree of elliptical polarization is determined. The polarizing plate 41 is rotated to a position where “substantially 0” is obtained. Then, the driving units 51 and 52 rotate the first and second compensators 7 and 10 by the same amount as the rotation amount of the polarizing plate 41, thereby illuminating light L1 from the objective lens 4 and the condenser lens 6 and the like. The elliptical polarization of the observation light L2 can be corrected to maintain the linear polarization state of the illumination light L1 and the observation light L2. As a result, when illumination is performed with declination falling illumination and polarized light, illumination with a good polarization state can be realized, and fluorescence polarization observation can be performed with high contrast. Here, for the purpose of correction, the amount of rotation of the first compensator 7 and the amount of rotation of the second compensator 10 are set to the same amount because changes in the polarization state of illumination light and observation light are mainly the objective lens 4. This is because the amount of elliptical polarization for the illumination light L1 and the observation light L3 by the objective lens 4 is assumed to be substantially the same value. After measuring the amount of elliptical polarization and adjusting the positions of the first and second compensators 7, 10, the sample 30 is observed by returning the first mirror 17 to its original position.

  In the above embodiment, a typical polarizing microscope optical system has been described. However, the present invention is not limited to the polarization microscope, and it is possible to compensate for changes in polarization characteristics of various optical devices that use polarized light, such as ellipsometers and differential interference microscopes. Further, the above-described embodiment is merely an example, and is not limited to the above-described configuration, and can be appropriately changed within the effect range of the present invention. In the above description, the first compensator 7 corrects the elliptical polarization amount of the illumination light L1 in the illumination optical system 2, and the second compensator 10 corrects the elliptical polarization amount of the observation light L2 in the observation optical system 3. Although the correction has been performed, it is needless to say that only the first compensator 7 may be provided.

It is explanatory drawing which shows the structure of the optical system of the microscope apparatus of this invention. It is explanatory drawing for demonstrating the illumination light which permeate | transmits the area | region where NA is 1 or more, (a) shows the path | route of the illumination light when seeing an objective lens from the side, (b) shows an objective lens from the upper surface. Shows the path of the illumination light when viewed. It is explanatory drawing for demonstrating the principle of a 1st and 2nd compensator, Comprising: (a) shows the structure of the compensator of Babinet-Soleil, (b) shows the structure of the compensator of Brace-Kohler.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 4 Objective lens 4a Pupil surface 5 Polarizer 6 Condensing lens 7 1st compensator 8 Imaging lens 9 Analyzer 10 2nd compensator 30 Sample L1 Illumination light L2 Observation light 40 Illumination light detection part 50 Control part (elliptical polarization) State calculation part)
100 Microscope device

Claims (9)

A light source that emits illumination light;
An objective lens that irradiates the sample with the illumination light that passes through a position greater than a predetermined NA;
A polarizer disposed between the light source and the objective lens for converting the illumination light into a linearly polarized state;
A condenser lens that condenses the illumination light transmitted through the polarizer on the pupil plane of the objective lens;
An imaging lens that emits the sample, collects the observation light transmitted through the objective lens, and forms an image of the sample;
An analyzer disposed between the imaging lens and the image and disposed such that a plane of polarization of transmitted light is orthogonal to the polarizer;
The elliptical polarization of the illumination light by the optical element that is disposed between the condenser lens and the polarizer and through which the illumination light passes is corrected to maintain the linear polarization state of the illumination light that irradiates the sample. And a first compensator.   The microscope apparatus according to claim 1, wherein the illumination light is transmitted through a region having an NA of 1 or more in the objective lens. An illumination light detector that detects a polarization state of the illumination light transmitted through the objective lens;
An elliptical polarization state calculation unit that calculates the degree of elliptical polarization of the illumination light detected by the illumination light detection unit;
3. The microscope apparatus according to claim 1, wherein a phase difference is given to the illumination light by the first compensator so as to cancel out a deviation amount of the elliptical polarization of the illumination light calculated by the elliptical polarization state calculation unit. .   The microscope apparatus according to any one of claims 1 to 3, wherein the first compensator is configured by a Babinet-Soleil compensator.   The microscope apparatus according to claim 1, wherein the first compensator is a Brace-Kohler compensator.   The microscope apparatus according to claim 1, further comprising a second compensator that is disposed between the imaging lens and the analyzer and corrects elliptical polarization of the observation light by the objective lens.   The microscope apparatus according to claim 6, wherein the correction amount of the second compensator is the same as that of the first compensator.   The microscope apparatus according to claim 6 or 7, wherein the second compensator is a Babinet-Soleil compensator.   The microscope apparatus according to claim 6 or 7, wherein the second compensator comprises a Brace-Kohler compensator.

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