JP2012189659A - Focal point maintenance device and microscope apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a focal point maintenance device which detects only AF light with high sensitivity by reducing stray light and improves ability for maintaining a focal point, and to provide a microscope apparatus having the focal point maintenance device.SOLUTION: A focal point maintenance device 10 used in a microscope apparatus 40 comprises: a focus optical system 30 for irradiating a specimen with focus light emitted from a light source part 20 through an objective lens 41 and guiding the focus light reflected on the specimen to a QPD 50; and a control part 60 for maintaining a focal surface of the objective lens 41 at a predetermined position on the specimen by changing relative positional relation between the objective lens 41 and the specimen, based on a detection signal output from the QPD 50. An incident angle of the focus light to a reflection surface of the specimen is the Brewster angle.

Description

  The present invention relates to a focus maintaining apparatus and a microscope apparatus.

  In recent years, a microscope apparatus having a focus maintaining apparatus is expanding the market. A focus maintaining device that keeps focusing on the specimen at all times can effectively remove blurs and blurs of the observation image that accompany long-time time-lapse observation and reagent administration. One method for maintaining focus is an active method. This method detects the position of the specimen in the z-axis direction (the position of the objective lens in the optical axis direction) from the reflection of the focus light (hereinafter referred to as “AF light”) applied to the specimen (such as a cover glass) (for example, Patent Document 1). Specifically, the reflected light is detected by a photodetector, and position information in the z-axis direction is calculated from the light intensity distribution on the photodetector.

JP 2007-323094 A

  However, in the case of an active focus maintaining apparatus as described above, if unwanted light other than AF light (stray light) enters the photodetector, the light intensity distribution on the photodetector changes from the original shape of the AF light. However, there is a problem that it is difficult to calculate an accurate position in the z-axis direction. The cause of this stray light is reflection at the end face of the objective lens. This is because it is not easy to apply an antireflection film having a high reflectance to a broadband optical spectrum. Therefore, establishment of a method for removing stray light and achieving focus maintenance with high accuracy is required. For example, there is a method of reducing the influence of reflection from the central part of the objective lens by projecting a slit image and shielding the central part, but reflected light from the peripheral part of the objective lens is not considered.

  The present invention has been made in view of such problems, and is configured to detect only AF light with high sensitivity by reducing stray light in an active focus maintaining apparatus and to improve focus maintaining ability. An object of the present invention is to provide a focused focus maintaining apparatus and a microscope apparatus having the focused focus maintaining apparatus.

  In order to solve the above-described problem, a focus maintaining apparatus according to the present invention is a focus maintaining apparatus that maintains a focal plane of an objective lens of a microscope apparatus at a predetermined position of a specimen, and the focus maintaining apparatus emits focus light emitted from a light source unit. Based on the focus optical system that irradiates the sample through the objective lens and guides the focus light reflected by the sample to the light detection unit and the detection signal output from the light detection unit, the relative position between the objective lens and the sample And a control unit that maintains the focal plane of the objective lens at a predetermined position of the sample by changing the relationship, and the incident angle of the focus light with respect to the reflection surface of the sample is a Brewster angle .

  In such a focus maintaining apparatus, the light source unit includes a light source that emits focus light, a collimator lens that converts the focus light emitted from the light source into a substantially parallel light flux, and a mask in which an opening that allows substantially parallel light to pass is formed. It is preferable to have.

  In addition, such a focus maintaining device is used to form an incident height of the focus light from the optical axis at the pupil of the objective lens so that the incident angle of the focus light on the reflecting surface becomes a Brewster angle. It is preferable to set the height of the opening from the optical axis.

  In such a focus maintaining apparatus, it is preferable that the opening of the mask is formed in a circular shape, and a Gaussian filter is attached to the opening.

  Further, in such a focus maintaining apparatus, the focus optical system has a parallel plane plate, and the focus from the optical axis in the pupil of the objective lens is such that the incident angle of the focus light on the reflection surface becomes the Brewster angle. In order to form the incident height of light, it is preferable that the plane-parallel plate is disposed at a predetermined angle with respect to the chief ray of the focus light.

  In such a focus maintaining device, the light source unit includes a light source that emits focus light, a fiber that guides the focus light emitted from the light source, and a collimator that converts the focus light emitted from the fiber into a substantially parallel light flux. And a collimating lens of the fiber and the collimating lens to form an incident height of the focusing light from the optical axis at the pupil of the objective lens such that the incident angle of the focusing light on the reflecting surface is a Brewster angle. It is preferable to set the arrangement position.

  Further, in such a focus maintaining device, the light source unit includes a light source that emits focus light, a fiber that guides focus light emitted from the light source, and a collimator lens that converts the focus light emitted from the fiber into a substantially parallel light beam. A mirror that reflects the focus light emitted from the collimator lens and guides the focus light to the focus optical system, from the optical axis at the pupil of the objective lens such that the incident angle of the focus light on the reflection surface is the Brewster angle. In order to form the incident height of the focus light, it is preferable to set the mirror arrangement position.

  Moreover, the microscope apparatus according to the present invention includes any one of the above-described focus maintaining apparatuses.

  When the focus maintaining apparatus according to the present invention and the microscope apparatus having the focus maintaining apparatus are configured as described above, stray light can be reduced, and only AF light is detected with high sensitivity, thereby improving the ability of maintaining the focus. Can do.

It is explanatory drawing which shows the structure of the focus maintenance apparatus and microscope apparatus which concern on 1st Embodiment. It is explanatory drawing which shows the state of AF light in a sample surface, Comprising: (a) shows the polarization state of incident light, (b) shows the polarization state of AF light in a reflective surface, (c) is an objective lens. The beam profile of AF light with respect to the exit pupil of FIG. It is explanatory drawing which shows the polarization state of AF light. It is explanatory drawing which shows the relationship between the imaging surface of AF light by an objective lens, and a sample surface, Comprising: (a) shows the case where an imaging surface and a sample surface correspond, (b) shows a sample surface. The case where it exists in the heel side from an image formation surface is shown, (c) shows the case where a sample surface exists in the near side from an image formation surface. It is explanatory drawing which shows the beam profile of AF light in the exit pupil surface of an objective lens, Comprising: (a) shows the range in which AF light injects with a Brewster angle, (b) reflects only S-polarized AF light. The range is shown, and (c) shows the range for imparting the focus maintaining ability. It is explanatory drawing for demonstrating the light ray height of AF light in a mask position. It is explanatory drawing which shows the beam profile of AF light with respect to the exit pupil of an objective lens in 2nd Embodiment. It is explanatory drawing which shows the polarization state of AF light in 2nd Embodiment. It is explanatory drawing which shows the structure of the focus maintenance apparatus and microscope apparatus which concern on 3rd Embodiment.

[First Embodiment]
Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of the focus maintaining apparatus according to the present embodiment will be described. The focus maintaining apparatus 10 guides AF light from a light source unit 20 having a light source 21 such as an LED or LD to an objective lens 41 of a microscope apparatus 40 by using a focus optical system 30, and a specimen through the objective lens 41. The reflected light is radiated by a four-divided diode 50 (a photodetecting element in which a circular region is divided into four, and a PD (photodiode) is arranged in each region, hereinafter referred to as “QPD50”). The z-axis direction position of the specimen with respect to the objective lens 41 is detected by detecting with the configured light detection unit. In the following description, a surface that reflects AF light in order to maintain the focal plane of the objective lens 41 at a predetermined position of the sample is referred to as a “sample surface O”.

  The light source unit 20 of the focus maintaining apparatus 10 includes a light source 21, a collimating lens 22 that converts AF light (for example, infrared light) emitted from the light source 21 into a substantially parallel light beam, and a sample surface O of the AF light. The mask 23 changes the beam profile so that the incident angle with respect to becomes the Brewster angle. Note that an opening 23a is formed in the mask 23, and the light beam (beam profile) L0 that is substantially parallel by the collimator lens 22 is changed to a light beam (beam profile) L1 that is separated from the optical axis by a predetermined distance. It is configured to enter the focus optical system 30 along the optical axis of the objective lens 41.

  In addition, the focus optical system 30 includes, in order from the light source unit 20 side, a first polarizer 31 that converts AF light emitted from the light source unit 20 into linearly polarized light, and AF light that has passed through the first polarizer 31. AF light emitted from the light source unit 20, which is disposed on the image side of a half mirror 32 that transmits substantially half and reflects the rest, an offset lens 33 having a function to be described later, and an objective lens 41 that constitutes the microscope apparatus 40. And the dichroic mirror 34 that guides the AF light reflected by the sample surface O to the focus maintaining apparatus 10. In the microscope apparatus 40, the illumination light applied to the specimen surface O and the signal light emitted from the specimen surface O (for example, fluorescence excited by the illumination light) are transmitted through the dichroic mirror 34. Here, the signal light transmitted through the dichroic mirror 34 is guided to a second objective lens (not shown) to form an image of the sample. Further, between the dichroic mirror 34 and the half mirror 32 of the focus optical system 30 of the focus maintaining apparatus 10, the polarization axis of the AF light (reflected light) from the specimen surface O reflected by the dichroic mirror 34 is rotated. A λ / 2 plate 35 is disposed, and a second polarizer 36 and an imaging lens 37 are disposed in a direction in which the reflected light is reflected by the half mirror 32. The QPD 50 is disposed on the focal plane of the imaging lens 37. In addition to the QPD, a line CMOS, a line CCD, a PD array, or the like can be used for the light detection unit.

  In such a focus maintaining apparatus 10, AF light emitted from the light source 21 is converted into a substantially parallel light beam L 0 by the collimator lens 22, and then the light beam L 1 is separated from the optical axis by a predetermined distance by the mask 23. And enters the first polarizer 31. As shown in FIG. 3A, the light beam L1 transmitted through the first polarizer 31 is converted into linearly polarized light in the direction of 45 ° (the direction of vibration in the first and third quadrants). Thereafter, the light passes through the half mirror 32, undergoes a change in curvature by the offset lens 33, is further combined with illumination light by the dichroic mirror 34, and is guided to the objective lens 41. Then, the AF light condensed by the objective lens 41 is incident on the sample surface O at a Brewster angle. In the following description, the optical axis direction is the z-axis direction, the horizontal direction is the x-axis direction, and the vertical direction is the y-axis direction in a plane orthogonal to the z-axis direction.

  FIG. 2 shows how the AF light is reflected on the sample surface (reflection surface) O. FIG. By the first polarizer 31 disposed immediately after the light source unit 20, the light beam L1 linearly polarized in the direction of 45 ° with respect to each of the x axis and the y axis as shown in FIG. Is incident on. This polarization state can be considered as a combination of linearly polarized light in the x-axis direction and linearly polarized light in the y-axis direction having the same amplitude. When the light beam L1 is focused on the specimen surface O by the objective lens 41, as shown in FIG. 2B, the linearly polarized light in the x-axis direction corresponds to P-polarized light, and the linearly polarized light in the y-axis direction corresponds to S-polarized light. To do. As described above, the light beam L1 incident at the Brewster angle with respect to the sample surface O has a P-polarized light component due to reflection, so that the reflected light is S-polarized, that is, linearly polarized in the y-axis direction. (This light beam is L2.) The state of the light beam L2 is shown in FIGS. 2 (c) and 3 (b).

  The light beam L2 reflected by the sample surface O passes through the objective lens 41 again, is reflected by the dichroic mirror 34, passes through the offset lens 33, returns to a substantially parallel light beam, and further passes through the λ / 2 plate 35. As shown in FIG. 3E, the λ / 2 plate 35 has either a fast axis or a slow axis at an angle inclined 22.5 ° counterclockwise with respect to the vibration direction of the S-polarized light beam L2. The axes have been adjusted to match. As a result, the AF light is converted into 45 ° linearly polarized light that vibrates in the second and fourth quadrants of the xy coordinates (this light beam is denoted as L3). Here, the vibration direction of the light beam L3 transmitted through the λ / 2 plate 35 is orthogonal to the vibration direction of the light beam L1 after passing through the first polarizer 31 (FIG. 3C). The λ / 2 plate 35 preferably has an effective diameter substantially the same as the beam diameter. This is to prevent extra light (light beam L1 traveling from the light source unit 20 toward the sample surface O or light reflected by the objective lens 41) from entering the λ / 2 plate 35.

  The AF light (light beam L3) that has been changed in polarization state by the λ / 2 plate 35 is reflected by the half mirror 32 and passes through the second polarizer 36. At this time, since the transmission axis of the second polarizer 36 coincides with the polarization direction of the light beam L3, the light beam L3 is transmitted. On the other hand, the direction of polarization of stray light (for example, light reflected by the objective lens 41) substantially coincides with the light beam L1. For this reason, the transmission axis of the second polarizer 36 is orthogonal to the light beam L 1, and such stray light cannot pass through the second polarizer 36. Then, the light beam L <b> 4 that has passed through the second polarizer 36 is condensed on the line sensor 50 by the imaging lens 37. In this way, it is possible to remove stray light and detect only AF light (AF light reflected from the sample surface O) necessary for maintaining the focus with high sensitivity.

  The offset lens 33 has a function of shifting the AF light imaging position in the z-axis direction with respect to the focal plane of the objective lens 41. That is, when the AF light emitted from the light source unit 20 and made into a substantially parallel light beam by the collimator lens 22 is a substantially parallel light beam after passing through the offset lens 33, the position of the AF light imaged by the objective lens 41 is It substantially coincides with the focal plane of the objective lens 41. On the other hand, when the AF light emitted from the offset lens 33 is divergent light, the image formation position of the AF light moves to the back side (the direction away from the objective lens 41) from the focal plane of the objective lens 41, and the offset lens 33. When the AF light emitted from the light beam is convergent light, the image position of the AF light moves to the near side (direction approaching the objective lens 41) from the focal plane of the objective lens 41. In this way, by shifting (offset) the imaging position of the AF light in the optical axis direction with respect to the focal plane of the objective lens 41, the focus of the objective lens 41 is focused on a desired position in the sample. The focus can be maintained by reflecting the AF light at a position different from the focal plane of the objective lens 41 (for example, the boundary surface between the plate glass covering the specimen and the specimen (the above-described “specimen plane O”)).

  If the sample plane O is at the AF light imaging position defined by the offset lens 33, that is, if there is no defocus, the light beam L4 incident on the imaging lens 37 is parallel light as shown in FIG. And an image is formed at the center of the QPD 50. However, as shown in FIGS. 4B and 4C, when the focus shift occurs, the light intensity distribution on the QPD 50 changes. For example, as shown in FIG. 4B, when the specimen surface O and the objective lens 41 are separated, the light beam L4 incident on the imaging lens 37 becomes convergent light. Therefore, an image is formed in front of QPD 50 and shifted to the left on QPD 50. On the other hand, as shown in FIG. 4C, when the sample surface O and the objective lens 41 come closer, the light beam L4 incident on the imaging lens 37 becomes divergent light. For this reason, an image is formed behind the QPD 50 and shifted to the right on the QPD 50. In this way, based on the detection signal output from the QPD 50, the focus shift is detected as the image position shift on the image plane, and the objective lens 41 is controlled by the control unit 60 so as to maintain the state of FIG. Alternatively, focus maintenance can be achieved by moving the specimen in the optical axis direction and performing feedback control.

  In order to realize this method, the Brewster illumination mask 23 described above needs to have the shape shown in FIG. FIG. 5 shows the position of the opening 23a of the mask 23 in relation to the exit pupil plane P0 of the objective lens 41 and the diameter of the light beam L1 that has passed through the opening 23a. In the exit pupil plane P0 of the objective lens 41, the distribution of the AF light in which the incident angle of the AF light to the sample surface O becomes the Brewster angle is as shown in FIG. In this state, the polarization state of incident light and reflected light does not change due to the averaging effect. This is because there are many incident surfaces. In contrast, in FIG. 5B, there is only one incident surface (in the x-axis direction). The light oscillating in the x-axis direction is P-polarized light, and the light oscillating in the y-axis direction is S-polarized light. When this light is incident on the sample surface O at the Brewster angle, the reflected light is only S-polarized light. In order to further give the Brewster illumination a focus maintaining ability, an illumination pattern as shown in FIG. 5C is used.

Here, when a light beam traveling through a medium having a refractive index n ₁ is incident on an interface with the medium having a refractive index n ₂ , in order for the incident angle to become the Brewster angle θ _Brew , the following conditional expression (a) Need to be satisfied.

That is, in order for the AF light to enter the sample surface O while satisfying the Brewster angle condition, it depends on what interface the sample surface O is. For example, in the case of a dry objective lens, the interface between air (n ₁ = 1) and glass (n ₂ = 1.55) is the sample plane O. At this time, the Brewster angle θ _Brew is 57.17 °. On the other hand, if the interface between the glass (n ₁ = 1.55) and the cell / water (n ₂ = 1.33) is the sample surface O, the Brewster angle θ _Brew is 40.63 °. This angle is common to the immersion objective lens and the oil immersion objective lens when the same surface is the sample surface O. Therefore, in order for AF light to enter the specimen surface O at a Brewster angle, the objective lens 41 to be used needs to have a numerical aperture (NA) of at least 40.63 ° or more. Here, the maximum incident angle θ _max of the objective lens 41 is determined by the NA of the objective lens 41 and the refractive index n of the medium, and is expressed by the following equation (b).

If NA required for dry, liquid immersion and oil immersion objective lenses are NAD, NAW and NAO, respectively, it is necessary to satisfy the condition of NA> n × sin θ _Brew from the above formula (b), and NAD> 0.651. NAW> 0.866 and NAO> 1.009. However, the refractive index of the medium was n = 1 for the dry objective lens, n = 1.33 for the immersion objective lens, and n = 1.55 for the oil immersion objective lens.

  Further, the position of the opening 23a of the Brewster illumination mask 23 needs to be changed for each objective lens 41 to be used. The exit pupil diameter of the objective lens 41 varies greatly depending on the magnification, and generally decreases as the magnification decreases. Therefore, it is necessary to change the position of the light beam (AF light) that becomes the Brewster angle for each objective lens 41. For this purpose, the mask 23 stores an optimum beam position for each objective lens 41 in advance, and is automatically based on the NA, focal length, and magnification information of the objective lens 41 each time the objective lens 41 is replaced. The position of the opening 23a is stored and moved to an optimum position. The type of the objective lens 41 may be detected by the control unit 60, and the position of the opening 23a of the mask 23 may be automatically moved based on the information of the objective lens 41. The mask 23 provided in accordance with the above may be configured to be switched simultaneously with the replacement of the objective lens 41.

Here, the height h _mask from the optical axis of the opening 23a formed in the mask 23 for the AF light to satisfy the Brewster angle condition will be described with reference to FIG. As shown in FIG. 6, when the refractive indexes of the medium before and after the sample surface O are n ₁ and n ₂ , respectively, the maximum incident angle θmax of the objective lens 41 is expressed by the following equation (b ′) from the above equation (b). It is expressed as follows.

  On the other hand, the radius d of the exit pupil P0 of the objective lens 41 is expressed by the following equation (c) where the focal length of the objective lens 41 is fo.

Further, the ray height h _exit from the optical axis of the AF light at the exit pupil P0 of the objective lens 41 for the AF light to enter the sample surface O at the Brewster angle θ _Brew is the radius d and the maximum incident angle θ of the exit pupil P0. _When expressed by _max , the following equation (d) is obtained.

If the focal lengths of the two lenses constituting the offset lens 33 are f ₁ and f ₂ in this order from the light source side, the light after passing through the opening 23a with respect to the ray height h _exit from the optical axis after passing through the offset lens 33. A ratio ratio of the ray height h _mask from the axis is expressed by the following equation (e).

From the above, the light ray height in the mask 23, that is, the height h _mask from the optical axis of the opening 23a needs to satisfy the following expression (1).

  In the case of a focus maintaining apparatus that does not have the offset lens 33, this can be dealt with by omitting the term of the ratio ratio of the light beam height (1 in the equation (1)).

  Further, in order to allow only the light incident on the sample surface O at the Brewster angle to pass through the opening 23a of the mask 23, the size (diameter) of the opening 23a in the x-axis direction is preferably as small as possible. . Further, in order to obtain highly linear (linear) polarized light, the size (diameter) of the opening 23a in the y-axis direction is preferably as small as possible. Therefore, it is preferable to reduce the diameter of the opening 23a of the mask 23. In order to remove the influence of diffraction by the opening 23a, it is preferable to make the shape of the opening 23a circular and attach a Gaussian filter to the opening 23a. Further, it is desirable that the opening 23 a of the mask 23 is moved to an optimal position for each offset amount of the offset lens 33 based on the conditional expression (1).

  In addition, as a method for detecting the defocus, it is also possible to use a knife edge (half moon focusing) method in which the diameter of the parallel light flux from the light source 21 is approximately halved and projected onto the sample surface O. A half moon mask and Brewster are used. It is also possible to configure so that the illumination mask 23 can be switched.

[Second Embodiment]
In the first embodiment, in order to guide the AF light emitted from the light source unit 20 in the focus maintaining apparatus 10 to the sample surface O and to guide the AF light reflected by the sample surface O to the QPD 50, the focus optical system Although the case where the half mirror 32 is used for 30 has been described, a polarizing beam splitter can also be used.

  Assuming that the position of the transmission / reflection surface of the polarization beam splitter is the same as the position of the transmission / reflection surface of the half mirror 32 in FIG. 1, the transmission axis of the first polarizer 31 is in the y-axis direction described above. Are linearly polarized in a direction along the y-axis as shown in FIG. 8A (the vibration direction of the AF light is deviated 45 ° counterclockwise with respect to the case of the first embodiment). It is necessary to make the light beam L1 incident on the objective lens 41. Further, the position of the AF light (incident light beam L1 and reflected light beam L2) at the exit pupil P0 of the objective lens 41 also needs to be shifted by 45 ° counterclockwise. That is, the incident surface (a surface including the light beam L1 and the light beam L2 at P0 in FIG. 7 and parallel to the z axis) and the vibration direction of the incident light beam must form an angle of 45 °.

  Here, it can be considered that the light beam L1 is a combination of linearly polarized light in directions of ± 45 ° with respect to the y-axis having the same amplitude, and the light beam L1 is condensed on the sample surface O by the objective lens 41. At this time, as shown in FIGS. 7 and 8B, linearly polarized light in the 45 ° (+ 45 °) direction counterclockwise with respect to the y-axis is converted to S-polarized light and 45 ° clockwise with respect to the y-axis. Linearly polarized light in the (−45 °) direction corresponds to P-polarized light.

  Since the light beam L1 incident on the sample surface O at the Brewster angle has a zero P-polarized component due to reflection, the reflected light becomes S-polarized light (this light beam is L2). The state of the light beam L2 is shown in FIGS. 7 and 8B. The light beam L2 passes through the objective lens 41 again, is reflected by the dichroic mirror 34, passes through the offset lens 33, returns to a substantially parallel light beam, and further passes through the λ / 2 plate 35. As shown in FIG. 8E, the λ / 2 plate 35 has either a fast axis or a slow axis at an angle inclined by 22.5 ° counterclockwise with respect to the vibration direction of the S-polarized light beam L2. The axes have been adjusted to match. Thereby, the AF light is converted into linearly polarized light that vibrates in the x-axis direction (this light beam is denoted as L3). Here, the vibration direction of the light beam L3 transmitted through the λ / 2 plate 35 is orthogonal to the vibration direction of the light beam L1 after passing through the first polarizer 31 (FIG. 8C). The λ / 2 plate 35 preferably has an effective diameter substantially the same as the beam diameter. This is to prevent extra light (light beam L1 directed from the light source unit 20 toward the sample surface O or light reflected by the objective lens) from entering the λ / 2 plate 35.

  The AF light (light beam L3) that has been changed in polarization state by the λ / 2 plate 35 is reflected by the half mirror 32 and passes through the second polarizer 36. At this time, since the transmission axis of the second polarizer 36 coincides with the polarization direction of the light beam L3, the light beam L3 is transmitted. On the other hand, the direction of polarization of stray light (for example, light reflected by the objective lens 41) substantially coincides with the light beam L1. For this reason, the transmission axis of the second polarizer 36 is orthogonal to the light beam L 1, and such stray light cannot pass through the second polarizer 36.

[Third Embodiment]
In the focus maintaining apparatus 10 according to the first and second embodiments, the incident angle of the AF light with respect to the specimen surface O is set to the position of the opening 23a of the mask 23 (position in a plane orthogonal to the optical axis of the objective lens 41). However, as in the focus maintaining apparatus 100 shown in FIG. 9, the light emitted from the fiber 210 is used as a light source, and the fiber 210 is deviated with respect to the optical axis of the objective lens 41. By making the core, the incident angle of the AF light with respect to the specimen surface O can be changed. The light source unit 200 in the focus maintaining apparatus 100 includes a fiber 210 that guides light from a light source (not shown), a collimator lens 220 that converts AF light emitted from the exit end of the fiber 210 into a substantially parallel light beam, and a collimator lens 220. And a mirror 230 for allowing the transmitted AF light to enter the focus optical system 30 along the optical axis of the objective lens 41 (the same reference numerals are used for the same components as those in the focus maintaining apparatus 10 according to the first embodiment). And a detailed description is omitted). In the focus maintaining apparatus 100 shown in FIG. 9, instead of decentering the fiber 210 with respect to the optical axis of the objective lens 41, the height of the AF light with respect to the optical axis is changed by decentering the mirror 230. It is configured to be incident on the surface O at a Brewster angle. When the AF light emitted from the collimating lens 220 is directly incident on the focus optical system 30 without using the mirror 230, the exit end of the fiber 210 and the collimating lens 220 are integrated with the optical axis of the objective lens 41. To be eccentric.

  According to the focus maintaining apparatus 100 according to the third embodiment, the light source unit 200 does not need a mask. At this time, it is necessary to collimate using a collimating lens 220 having a short focal length so that the beam diameter does not become too large.

  In addition to the first to third embodiments, in order to make the AF light emitted from the light source unit 20 incident on the sample surface O at the Brewster angle, the focus described in the first embodiment. AF light emitted from the light source unit between the first polarizer 31 and the dichroic mirror 34 of the focusing optical system 30 of the maintenance device 10 (more preferably, between the first polarizer 31 and the offset lens 33). A plane parallel plate may be arranged so as to form a predetermined angle with respect to the principal ray. By changing the angle formed between the principal ray of the AF light and the plane parallel plate, the height of the AF light emitted from the plane parallel plate with respect to the optical axis can be changed.

  As described above, by configuring the AF light to be incident on the sample surface O at the Brewster angle, only the signal intensity of the AF light necessary for maintaining the focus (AF light reflected from the sample surface O) is increased. Since it can detect with sensitivity, the maintenance system of the focus position of the objective lens 41 by the focus maintenance apparatuses 10 and 100 can be improved.

DESCRIPTION OF SYMBOLS 10 Focus maintenance apparatus 20 Light source part 21 Light source 22 Collimating lens 23 Mask 23a Opening part 30 Focus optical system 40 Microscope apparatus 41 Objective lens 50 QPD (light detection part) 60 Control part 100 Focus maintenance apparatus 200 Light source part 210 Fiber 220 Collimating lens 230 mirror

Claims (8)

A focus maintaining device that maintains a focal plane of an objective lens of a microscope apparatus at a predetermined position of a specimen,
A focus optical system that irradiates the specimen with focus light emitted from a light source section via the objective lens, and that guides the focus light reflected by the specimen to a light detection section;
A control unit that maintains a focal plane of the objective lens at a predetermined position of the sample by changing a relative positional relationship between the objective lens and the sample based on a detection signal output from the light detection unit; Have
The focus maintaining apparatus, wherein an incident angle of the focus light with respect to a reflection surface of the specimen is a Brewster angle. The light source unit is
A light source that emits the focus light;
A collimating lens for converting the focus light emitted from the light source into a substantially parallel luminous flux;
The focus maintaining apparatus according to claim 1, further comprising: a mask having an opening through which the substantially parallel light passes.   In order to form an incident height of the focus light from the optical axis at the pupil of the objective lens such that an incident angle of the focus light to the reflection surface is a Brewster angle, The focus maintaining apparatus according to claim 2, wherein a height from the optical axis is set.   The focus maintaining apparatus according to claim 2, wherein the opening of the mask is formed in a circular shape, and a Gaussian filter is attached to the opening. The focus optical system has a plane parallel plate,
In order to form an incident height of the focus light from the optical axis at the pupil of the objective lens such that an incident angle of the focus light on the reflection surface becomes a Brewster angle, The focus maintaining apparatus according to claim 2, wherein the plane-parallel plate is disposed at a predetermined angle with respect to the focus maintaining apparatus. The light source unit is
A light source that emits the focus light;
A fiber for guiding the focus light emitted from the light source;
A collimating lens that makes the focus light emitted from the fiber a substantially parallel light beam,
In order to form an incident height of the focus light from the optical axis at the pupil of the objective lens such that an incident angle of the focus light on the reflection surface becomes a Brewster angle, the fibers and the collimating lens The focus maintaining apparatus according to claim 1, wherein an arrangement position is set. The light source unit is
A light source that emits focus light;
A fiber for guiding the focus light emitted from the light source;
A collimating lens for converting the focus light emitted from the fiber into a substantially parallel luminous flux;
A mirror that reflects the focus light emitted from the collimator lens and guides the focus light to the focus optical system,
The arrangement position of the mirror is set in order to form an incident height of the focus light from the optical axis at the pupil of the objective lens so that an incident angle of the focus light on the reflection surface becomes a Brewster angle. The focus maintaining apparatus according to claim 1, wherein:   A microscope apparatus comprising the focus maintaining apparatus according to claim 1.

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