JP2011059205A - Microscope device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microscope device capable of obtaining a good image with less aberration, even when the position of a pupil conjugate surface is fluctuated in the case of observing a phase object by relaying a pupil of an object lens and arranging a pupil modulation element on the pupil conjugate surface. <P>SOLUTION: The microscope device includes: the objective lens 10 for emitting parallel luminous flux based on light from a sample 8; an imaging lens 20 for forming the image of the sample 8 based on the parallel luminous flux from the objective lens 10; and a relay optical system 40 for relaying the image. The relay optical system 40 includes a first relay lens group 41 and the second relay lens group 42, and the pupil conjugate surface 29 which is conjugate to the pupil 11 of the objective lens 10 is disposed between the first relay lens group 41 and the second relay lens group 42. When an angle formed by a principal light incident on the imaging lens 20 and the optical axis is denoted as θ, a composite focal length is denoted as f, and a distance of an image formed on the position conjugate to the pupil position of the objective lens, from the optical axis is denoted as y, the microscope device satisfies a conditional expression of f×tanθ>y>f×θ. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a microscope apparatus.

  An observation method using a pupil modulation element, such as phase difference observation, is known as a method for observing cultured cells alive without performing treatments such as solid and antibody staining when observing cultured cells. In such an observation method, it is necessary to use an objective lens in which a pupil modulation element such as a phase film is arranged in an internal pupil. However, a microscope apparatus capable of observing a phase difference without using this type of objective lens is also well known. A primary image formed by an imaging lens is relayed by a relay optical system, and the objective lens There is a microscope apparatus in which an exit pupil image is disposed between relay optical systems and a pupil modulation element is disposed at the position of the pupil image, thereby eliminating the need for the objective lens as described above (for example, see Patent Document 1). reference).

Japanese Patent Application Laid-Open No. 08-15612

  However, in the microscope apparatus as described above, when the position of the pupil fluctuates due to switching or movement of the objective lens, a lens disposed between the pupil conjugate plane and the pupil conjugate plane in order to arrange the phase film on the pupil conjugate plane. A different aberration occurs for each pupil plane. When different aberrations occur for each pupil plane, depending on how the aberrations appear, there is a problem that the image of the ring diaphragm arranged at the pupil position of the condenser lens and the phase film may be misaligned, and the phase difference image cannot be observed well. there were.

  The present invention has been made in view of such a problem, and the position of a pupil conjugate plane is observed when a phase object is observed by relaying the pupil of an objective lens and arranging a pupil modulation element on the pupil conjugate plane. An object of the present invention is to provide a microscope apparatus capable of obtaining a good image with little occurrence of aberrations even when fluctuates.

を満足することを特徴とする。 In order to achieve such an object, a microscope apparatus according to the present invention includes an illumination unit that illuminates a specimen with illumination light, an objective lens that collects light emitted from the specimen by illumination of the illumination unit, and an objective lens. An image forming lens that forms an image of the sample by using and a relay lens group that relays the image formed by the image forming lens are provided. The relay lens group includes a first relay lens group and a second relay lens group. In a microscope apparatus in which a position conjugate with the pupil position of the objective lens with respect to the imaging lens and the first relay lens group is provided between the first relay lens group and the second relay lens group, θ is imaged The angle between the principal ray incident on the lens and the optical axis of the imaging lens and the first relay lens group, f the combined focal length of the imaging lens and the first relay lens group, and y the pupil position of the objective lens Together with When the height from the optical axis of the image formed on the Do position, condition

It is characterized by satisfying.

In the above-described microscope apparatus, it is preferable that the imaging lens and the first relay lens group satisfy the following conditional expressions.

  Further, the imaging lens and the first relay lens group are image-side telecentric, and a light modulation element that modulates the light that has passed through the first relay lens group is provided at a position conjugate with the pupil position of the objective lens. Preferably, the imaging lens or a part of the lenses constituting the first relay lens group, or the light modulation element is configured to be movable in the optical axis direction of the imaging lens and the first relay lens group. It is preferable that the element is a phase modulation element that phase modulates light.

  According to the present invention, when observing a sample such as a phase object, even if the position of the pupil conjugate plane provided at a position conjugate with the pupil position of the objective lens fluctuates due to switching of the objective lens, etc., aberration is generated. A good image with less can be obtained.

It is the schematic which shows an example of the microscope apparatus which concerns on this invention. It is an optical path diagram for demonstrating the projection system of the lens group which is the basic composition of this invention.

  Hereinafter, although this embodiment is described, referring to drawings, the following embodiment does not limit the invention concerning a claim. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention.

  The microscope apparatus according to the present embodiment is a microscope apparatus capable of phase difference observation that can be observed alive without performing processing such as cell fixation and antibody staining when observing cultured cells. As shown in FIG. 1, this microscope apparatus includes a light source device 1 for transmission, a collector lens 2, a field stop 3, a mirror 4, a lens group 5, an aperture stop 6, a condenser lens 7, and a stage 9 on which a specimen 8 is placed. It is configured with. The light emitted from the transmission illumination light source 1 is converted into a parallel light beam by the collector lens 2, passes through the field stop 3, is deflected downward by the mirror 4, and is condensed on the aperture stop 6 by the lens group 5. . The light collected by the aperture stop 6 is converted into a parallel light beam by the condenser lens 7 and then illuminates the specimen 8 placed on the stage 9. When performing phase difference observation, a ring stop is disposed at a position corresponding to the aperture stop 6 so that the sample is illuminated only with a predetermined light beam. An image of the ring diaphragm is formed on a pupil conjugate plane, which will be described later, and the intensity and phase of illumination light are modulated by a pupil modulation element (phase modulation element) in which a phase film is provided in a region corresponding to the aperture of the ring diaphragm. Since ring diaphragms are often roughly classified into 3 to 4 ring diameters depending on the NA of the objective lens, ring diaphragms with different ring diameters can be placed in a turret (not shown) and selectively inserted into the optical path. Is possible.

  An objective lens 10, an imaging lens 20, a prism 21, an imaging element 22 and a mirror 23 are provided below the sample 8, and a pupil 11 that is an exit pupil is provided inside the objective lens 10. . Each member from the light source device for transmission 1 to the objective lens 10 is configured to be movable in the direction of the optical axis (also referred to as stage up since the height of the specimen stage is set to a high position). 10 and the imaging lens 20 can be adjusted (detailed later). The objective lens 10 is attached to a revolver (not shown) and can be switched to a different objective lens by rotating the revolver.

  The light transmitted through the specimen 8 enters the objective lens 10, passes through the pupil 11 inside the objective lens 10, and a primary image 24 is formed by the imaging lens 20. The prism 21 is provided behind the imaging lens 20, and the prism 21 forms a primary image on the upper part of the image sensor 22 so that an image of the specimen 8 can be obtained.

  The microscope apparatus is provided with a filter block composed of a dichroic mirror 12, an excitation filter 13, and a fluorescent filter 14, and an epi-illumination light source 15. An interval of a predetermined length is provided between the objective lens 10 and the imaging lens 20, and a filter block is provided at this interval, and light emitted from the epi-illumination light source 15 passes through this filter block. The sample 8 is irradiated from below. Although FIG. 1 shows an example in which only one filter block is provided, since the objective lens 10 and the imaging lens 20 are in a parallel system, imaging with the objective lens 10 is performed by stage-up. Two filter blocks can be inserted with the distance between the lenses 20 widened. It is also possible to illuminate the specimen 8 by making light having different wavelengths incident as excitation light via these filter blocks. In the microscope apparatus used in this embodiment, the primary image 24 is formed at a position conjugate with the sample 8 with respect to the objective lens 10 and the imaging lens 20, but when the filter block is inserted in the optical path. Is formed so as to pass through the filter block and the imaging lens 20 and be deflected laterally by the mirror 23. Also, the filter block is housed in the turret, and insertion / removal to the optical path is performed by rotation of the turret, but there is a hole in the turret position, and the filter block is out of the optical path at the hole position, Only the imaging light by transmitted illumination can pass through.

  Further, the microscope apparatus according to the present embodiment includes a relay optical system 40, and the relay optical system 40 includes a first relay lens group 41 and a second relay lens group 42. The first relay lens group 41 includes a first lens group 25, a mirror 26, a second lens group 27, and a third lens group 28, and the second relay lens group 42 includes the fourth lens group 31 and the third lens group 28. And 5 lens groups 32. Each lens group is configured by combining a convex lens and a concave lens. The light forming the primary image 24 is relayed by the first relay lens group 41 so as to pass through the first lens group 25, the mirror 26, the second lens group 27, and the third lens group 28. The configuration of each lens group of the relay optical system 40 is not limited to the above. That is, the configuration can be appropriately changed. For example, the first lens group 25 can be positioned at the rear stage of the mirror 26.

  The first relay lens group 41 and the second relay lens group 42 are in a parallel system. As shown in FIG. 1, no connection is made between the first relay lens group 41 and the second relay lens group 42. A pupil conjugate plane 29 that is conjugate with the pupil 11 with respect to the image lens 20 and the first relay lens group 41 is formed. Therefore, even if no pupil modulation element is arranged on the pupil 11 inside the objective lens 10, a pupil modulation element (phase modulation element) is arranged on the pupil conjugate plane 29 and a pupil-modulated image can be observed. It can be done. In the present embodiment, for example, a mirror 30, a lens 33, and an image sensor 34 are provided behind the pupil conjugate plane 29, and an image is formed on the image sensor 34 on the lens 33 using light reflected by the mirror 30. By doing so, it is possible to obtain a pupil-modulated image. A mirror 35 and an eyepiece lens 37 are provided at the subsequent stage of the relay optical system 40, and the light relayed by the relay optical system 40 is deflected by the mirror 35 and then forms a secondary image 36. ing. The secondary image 36 is formed at a position conjugate with the primary image 24 with respect to the relay optical system 40, and this is observed as an image of the specimen 8 by the observer's observation eye 38 through the eyepiece 37. Yes.

  As described above, in the microscope apparatus of the present embodiment, the primary image 24 imaged by the imaging lens 20 is relayed by the relay optical system 40, the pupil conjugate plane 29 of the pupil 11 is disposed between the relay optical system 40, It is possible to adopt a configuration in which a pupil modulation element is arranged at the position of the pupil conjugate plane 29. By the way, in this type of microscope apparatus, for example, even if the pupil conjugate plane has no aberration when a relay lens group is designed assuming a certain objective lens, it can be replaced with a different objective lens or staged up. When the object distance (here, the distance between the pupil of the objective lens and the imaging lens) changes, aberration occurs in the image formed on the pupil conjugate plane. And depending on how this aberration appears, the position and size of the phase diaphragm in the pupil modulation element and the image of the ring diaphragm arranged on the pupil plane of the condenser lens are shifted, resulting in more light and lowering the contrast, Color unevenness occurs and the phase difference image cannot be observed well. Further, since the relay optical system of the microscope apparatus generally has a large F-number (dark) of the lens, this aberration is mainly a field curvature. Therefore, it is necessary to reduce the influence of this field curvature. Further, even if the position of the pupil conjugate plane 29 changes, the aperture of the subsequent lens does not become too large, and the pupil image size does not change even if the pupil modulation element moves during focusing. It is desirable that the principal ray is telecentric before and after the surface 29 and is parallel to the optical axis.

  Therefore, the relay lens group in the microscope apparatus of the present embodiment can reduce the influence of field curvature using the following projection method. The projection method will be described with reference to FIG. In FIG. 2, a main plane 100, a front focal plane 101, a rear focal plane 102, an object plane 103, and an image plane 104 are shown. Here, the object plane 103 is the pupil 11 of the objective lens 10 in FIG. 1, the lens group L in the main plane 100 is the imaging lens 20 to the second convex lens 28 (first relay lens group 41) in FIG. The image plane 104 corresponds to the pupil conjugate plane 29 in FIG. 1, that is, the plane into which the pupil modulation element (phase modulation element) is inserted. In FIG. 2, it is assumed that light travels from the left side (designed object side) to the right side (designed image side) as in a normal optical design.

Here, when the focal length of the lens unit L is f, the front focal plane 101 exists at a position separated by f on the object side with respect to the main plane 100, and the rear focal plane 102 exists at a position separated by f on the image side. To do. In this embodiment, a telecentric case is considered. That is, the stop 105 is virtually provided on the front focal plane 101 (in an actual microscope apparatus, the primary image 24 corresponds to this position). By doing so, the imaging principal ray becomes parallel to the optical axis on the right side of the lens unit L, and a so-called image-side telecentric optical system is configured. Further, the rear focal plane 102 has a conjugate relationship with the infinity distance, and an image at infinity is formed on the rear focal plane 102. The object plane 103 is located on the front side by L ₀ from the front focal plane 101. Note that the object plane 103 and the image plane 104 have a conjugate relationship. For example, the object point 110 located on the object plane 103 in a direction extending in a direction perpendicular to the optical axis Ax from the optical axis Ax is on the image plane 104. An image is formed at an image point 111 located at a location extending from the optical axis Ax in the direction opposite to the above direction. Further, the object point 120 on the optical axis Ax on the object plane 103 forms an image on the image point 121 on the optical axis Ax on the image plane 104. At this time, the distance between the rear focal plane 102 and the image plane 104 is x ′.

Hereinafter, a method for reducing the influence of the above-described field curvature will be considered. First, even if the object moves in the optical axis direction, the Betzval sum, which is a unique value of the lens group L, does not change. Therefore, sagittal field curvature can be reduced if the occurrence of meridional field curvature is suppressed. Therefore, the projection relationship of the lens group L is expressed by the equation (1).

Differentiating both sides of Equation (1) gives Equation (2).

Moreover, Formula (3) is guide | induced from the geometric relationship in FIG. In Expression (3), “a” is the radius of the diaphragm 105.

In order to prevent curvature of field from occurring even when the object moves in the optical axis direction, the movement of the paraxial image point position due to the object movement and the movement of the memorial image point off the axis need only coincide. . Specifically, it may be present on the image plane 104 that is a plane on which the image point 121 with respect to the object point 120 on the optical axis Ax of the object plane 103 exists. In general, the image plane movement x ′ on the optical axis Ax can be obtained from Equation (4) from Newton's formula.

On the other hand, if it is assumed that the image point 111 exists on the image plane 104, when the opening angle on the image plane is α and the change in image height when the chief ray is inclined by Δθ is Δy, Equation (5) is established. .

Here, since a very thin light beam is considered as the memorial image point, tan α≈sin α is set, and when equation (5) is modified in accordance with this, equation (6) is obtained.

Further, from the relation corresponding to Helmholtz-Lagrange, when the Strobel's theorem is applied in the meridional plane, Equation (7) is derived.

As described above, the function g can be obtained using the equations (2) to (7). First, equation (8) is obtained from equation (7).

By substituting equation (8) into equation (6), equation (9) is obtained.

Further, when Expression (2) and Expression (4) are substituted into Expression (9), Expression (10) is derived.

Further, when Expression (3) is substituted into Expression (10), Expression (11) is obtained.

When equation (11) is integrated, equation (12) is obtained. In addition, F in Formula (12) is 1st type elliptic integral.

In order to facilitate the solution of Equation (12), Equation (13) is obtained by substituting an approximate equation into Equation (11) and solving.

Therefore, the equation (14) is obtained by integrating the equation (13).

  From the above, the function g was obtained as the equation (12), and the result expressed approximately by the equation (14) was obtained. In other words, if the lens group L is designed so as to have a projection relationship obtained by substituting the function g into the expression (1), an optical system free from curvature of field in any plane on the image side can be realized. it can.

Next, the relationship between the optical system having the above projection relationship will be considered. In a general ftan θ lens, the function g is as shown in Expression (15).

Further, in a general Fourier transform lens (fsin θ lens), the function g is expressed as in Expression (16).

Furthermore, in a general fθ lens, the function g is expressed as in Expression (17).

  Comparing Expression (15), Expression (16), and Expression (14), the projection relationship of the lens unit L in this embodiment is a projection relationship between the projection relationship of the ftan θ lens and the projection relationship of the fθ lens. I can say that. Within this range, it is possible to realize an optical system with extremely little curvature of field in any plane on the image side.

  The microscope apparatus according to the present embodiment including the optical system configured as described above includes, for example, one or both of a ring stop of a condenser lens and a pupil modulation element to be inserted into an imaging system as an SLM (Spatial Light Modulator, spatial light). When configured with an optical element that can electrically change the ring diameter and phase modulation area, such as a modulator, the image plane (pupil conjugate plane) will shift even if the ring diameter is changed because the curvature of field is small. There is no effect.

Further, the fact that the position of the pupil differs by switching the objective lens or raising the stage corresponds to a change in L ₀ in FIG. When L ₀ changes in this way, it is necessary to move the pupil modulation element or lens group L to the corresponding position. However, since the field curvature is small, the amount of movement depends on the ring diameter of the pupil modulation element. Therefore, it is possible to easily determine the position, and further, it is possible to obtain an effect that fine adjustment of the position in consideration of the curvature of field is unnecessary.

DESCRIPTION OF SYMBOLS 1 Transmission light source device (illumination part) 8 Sample 10 Objective lens 20 Imaging lens 24 Primary image (image)
29 Pupil conjugate image (light modulation element, phase modulation element)
40 Relay optical system (relay lens group)
41 First relay lens group 42 Second relay lens group

Claims (6)

An illumination unit that illuminates the specimen with illumination light;
An objective lens that collects the light emitted from the specimen by illumination of the illumination unit and emits a parallel light beam;
An imaging lens that forms an image of the sample by injecting a parallel light beam emitted from the objective lens;
A relay lens group that relays an image formed by the imaging lens;
The relay lens group includes a first relay lens group and a second relay lens group, and the imaging lens and the first relay lens group are disposed between the first relay lens group and the second relay lens group. In a microscope apparatus in which a position conjugate with the pupil position of the objective lens is provided,
θ is an angle between the principal ray incident on the imaging lens and the optical axis of the imaging lens and the first relay lens group, and f is a combined focal length of the imaging lens and the first relay lens group. And y is the distance from the optical axis of the image formed at a position conjugate with the pupil position of the objective lens,

A microscope apparatus satisfying the following conditions. The imaging lens and the first relay lens group are:

The microscope apparatus according to claim 1, wherein the following condition is satisfied.   The microscope apparatus according to claim 1, wherein the imaging lens and the first relay lens group are image side telecentric.   The microscope apparatus according to claim 1, wherein a light modulation element that modulates light that has passed through the first relay lens group is provided at a position conjugate with a pupil position of the objective lens.   The imaging lens or a part of the lenses constituting the first relay lens group, or the light modulation element is configured to be movable in the optical axis direction of the imaging lens and the first relay lens group. The microscope apparatus according to claim 1, wherein the microscope apparatus is characterized.   The microscope apparatus according to claim 4, wherein the light modulation element is a phase modulation element that performs phase modulation of light.

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