JP2011008188A - Optical microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical microscope capable of performing observation by proper focal illumination by relatively rotating a sample and focal illumination with respect to the vertical axis of the sample, while being made to correspond to the shape of the sample, thereby performing irradiation, without intercepting the focal illumination by the sample.SOLUTION: The optical microscope includes a stage 2 holding the sample 3 and a transmission illumination optical system 4 illuminating the sample 3. The transmission illumination optical system 4 includes a light shielding means 43 asymmetrically intercepting a luminous flux from a light source 1, to perform illumination and the light shielding means 43 is configured so as to relatively rotate around an optical axis with respect to the stage 2.

Description

  The present invention relates to a transmission illumination optical system of a microscope, and more particularly to an optical microscope having an illumination optical system that irradiates a specimen with a light beam asymmetrically with respect to an optical axis.

  In recent years, cell analysis techniques for capturing large amounts of cultured cell images, extracting cell features from the obtained images using computer software, evaluating cell activity, and evaluating the effectiveness of drugs have become popular. It came to be done. In many cases, cells are subjected to experiments by labeling the cellular components (cell nucleus, membrane, Golgi apparatus, mitochondria, ER, or the entire cytoplasm) with a fluorescent reagent according to the purpose of analysis.

  However, the fluorescent reagent is basically toxic, and time-lapse observation in which images are taken intermittently in a state where cells are utilized, that is, while culturing, is basically impossible. As a technique for realizing this, a method of introducing a fluorescent protein into a cell has recently been actively performed. With the introduction of this technology, it has become possible to observe cells for a long time using a fluorescence microscope while visualizing cells.

  However, iPS cells and many stem cells that have been actively studied in recent years are cells with a low differentiation level, and gene transfer of fluorescent protein is originally a cell even though there is no chemical toxicity like a fluorescent reagent. There is no denying the possibility of affecting the characteristics of the. Therefore, for long-term observation of these cells, it has been strongly required to obtain image data over a long period of time by culturing the fluorescent protein as it is under a microscope without introducing a gene. In other words, it is desired that the above-described analysis can be performed from an image obtained by a transmission observation method such as a phase difference observation method, instead of relying on a fluorescent label.

However, the transmission observation method has the following drawbacks.
Usually, an inverted microscope is used to observe cells under a microscope while culturing cells. In order to culture cells, at least a culture solution is required, and a container for holding the culture solution, such as a petri dish or a microplate with containers arranged in an array, is used. Of course, an inverted microscope having an objective lens below the container is advantageous in terms of its construction.

  However, in the inverted microscope, the light transmitted from the condenser lens for the transmission illumination for obtaining the transmission image has a condensing angle, so that the illumination light hits the wall of the culture vessel and the illumination with sufficient performance cannot be obtained. Furthermore, a larger angle (NA) is required to increase the resolution of the microscope.

  Therefore, there is a method called oblique illumination as a means for solving the above-mentioned drawbacks in the transmitted illumination method. This is generally known as a technique for partially blocking the light beam and irradiating the sample with a light beam that is biased with respect to the optical axis, mainly to improve the contrast of the image. Note that “oblique illumination” is sometimes referred to as “oblique illumination”, “tilt illumination”, or “oblique illumination”, but is referred to as “oblique illumination” in the present application.

  And as an illuminating device that performs oblique illumination used in a microscope, for example, as shown in Patent Document 1, the light shielding sheet is inserted into and removed from the illumination optical path limited by the aperture stop, and the light shielding sheet is inserted into the illumination optical path. Some have a light shielding mechanism that shields the illumination light beam asymmetrically with respect to the illumination optical axis in accordance with the amount.

JP 2009-14472 A

  However, in the method shown in Patent Document 1 above, oblique illumination can be performed only from a certain direction on the specimen.For example, when a dish or a well plate is used as a specimen generally used in a microscope, Depending on the observation position, there is a problem that the wall for holding the culture medium of the sample blocks light and the oblique illumination cannot be performed appropriately.

  The present invention has been made in view of the above-described problems, and according to the shape of the specimen, the specimen and the oblique illumination are rotated relative to the vertical axis of the specimen so that the oblique illumination is performed by the specimen. An object of the present invention is to provide an optical microscope that is irradiated without being blocked and can be observed with appropriate oblique illumination.

  In order to achieve the above object, an optical microscope according to the present invention is an optical microscope including a stage that holds a specimen and a transmission illumination optical system that illuminates the specimen, and the transmission illumination optical system includes a light source. A light shielding means for asymmetrically shielding and illuminating the light flux, and the light shielding means is configured to rotate relative to the stage about the optical axis.

  In the optical microscope of the present invention, it is preferable that the rotational position of the light shielding means is automatically controlled in correspondence with the specimen based on the position information of the stage.

  In the optical microscope of the present invention, it is preferable that the light shielding unit is configured so that the shape of the light beam that has passed through the light shielding unit is a fan shape.

  Alternatively, the optical microscope of the present invention is an optical microscope including a stage that holds a specimen and a transmission illumination optical system that illuminates the specimen, and the transmission illumination optical system has a luminous flux that is asymmetric with respect to the optical axis. The light source is provided so that it can be lit, and the light source is configured so that the light beam rotates relative to the optical axis or the lighting position is selected. .

  In the optical microscope of the present invention, it is preferable that the rotational position or lighting position of the light source is automatically controlled in correspondence with the specimen based on the position information of the stage.

  In the optical microscope of the present invention, it is preferable that the light source is constituted by an array including a plurality of light sources.

  Furthermore, in the optical microscope of the present invention, it is preferable that the specimen held on the stage is a well plate or a dish-shaped culture container capable of holding a culture solution.

  In the optical microscope of the present invention, it is preferable that the transmission illumination optical system is applied to phase difference observation.

  According to the optical microscope of the present invention, the oblique illumination is not blocked by the specimen by rotating the specimen and the oblique illumination relative to the specimen's vertical axis in accordance with the shape of the specimen. And has the effect of being able to observe with appropriate oblique illumination.

It is a front view which shows the outline of Example 1 of the optical microscope which concerns on this invention. 6 is a plan view showing an example of a blocking plate of Example 2. FIG. An example of a culture container is shown, (A) is a top view, (B) is a front view. It is explanatory drawing which shows the rotation position of the shielding board shown in FIG. An example of a microplate is shown, (A) is a top view, (B) is a front view. It is a top view which shows the other example of the interruption | blocking board of Example 2. FIG. It is explanatory drawing which shows the slide position of the slider of Example 2. FIG. It is explanatory drawing which shows the rotational position of the slider of Example 2. FIG. (A) is a front view which shows the outline of Example 3 of the optical microscope which concerns on this invention, (B) is the example arrange | positioned so that the array which consists of several LED of Example 3 may circle | circulate on a concentric circle. It is a top view to show, (C) is a top view which shows the example by which the array which consists of several LED is arrange | positioned at a part on a concentric circle. It is explanatory drawing which shows the lighting position of LED shown in FIG.9 (B).

Prior to the description of the embodiments, the outline and effects of the present invention are described.
The optical microscope of the present invention is an optical microscope provided with a stage for holding a specimen and a transmission illumination optical system for illuminating the specimen, and the transmission illumination optical system shields the light beam from the light source by asymmetrically shielding the light. The light shielding means is configured to rotate relative to the stage about the optical axis.

  A rotation mechanism is provided that can rotate the stage on which the sample is placed relative to the means for illuminating the stage with asymmetric light shielding. Alternatively, a mechanism is provided that allows the means for illuminating the fixed specimen with asymmetric light shielding to be rotated with respect to the optical axis. By doing so, oblique illumination can be performed from a direction that does not block light, and observation with appropriate oblique illumination is possible.

  In the optical microscope of the present invention, it is preferable that the rotational position of the light shielding means is automatically controlled in correspondence with the sample based on the position information of the stage.

  In the case of an apparatus that performs automatic observation, the stage is rotated around the optical axis in accordance with the observation position of the specimen. Alternatively, the means for illuminating with asymmetric light shielding is adjusted to the observation position of the sample and rotated about the optical axis. As a result, the rotational position of the light shielding means is automatically adjusted according to the observation position of the specimen, and illumination can always be performed from a direction in which light is not obstructed by the specimen, thereby enabling automatic observation.

  In the optical microscope of the present invention, preferably, the light shielding means is configured such that the shape of the light beam that has passed through the light shielding means is a fan shape.

  When the objective lens is a phase difference objective lens, the shape of the light beam that has passed through the light shielding means is made to be a fan shape, and the light beam is projected onto the phase film of the objective lens. Thereby, phase difference observation becomes possible.

  Furthermore, the optical microscope of the present invention is an optical microscope including a stage that holds a specimen and a transmission illumination optical system that illuminates the specimen, and the transmission illumination optical system is configured such that the light beam is asymmetric with respect to the optical axis. A light source is provided so as to be lit, and the light source is configured such that the light beam rotates relative to the optical axis or the lighting position is selected.

  A rotation mechanism is provided that can rotate the stage on which the sample is placed relative to the light source that is lit so that the luminous flux is asymmetric with respect to the optical axis. Alternatively, the light source is configured so that the light beam can rotate about the optical axis or the lighting position can be selected with respect to the fixed specimen. By doing so, oblique illumination can be performed from a direction that does not block light, and observation with appropriate oblique illumination is possible.

  In the optical microscope of the present invention, preferably, the rotational position or lighting position of the light source is automatically controlled in correspondence with the sample based on the position information of the stage.

  In the case of an apparatus that performs automatic observation, the stage is rotated around the optical axis in accordance with the observation position of the specimen. Alternatively, the light source to be illuminated asymmetrically is aligned with the observation position of the sample and rotated around the optical axis, or the lighting position is appropriately selected. As a result, the rotation position or lighting position of the light source is automatically adjusted according to the observation position of the specimen, and illumination can always be performed from a direction in which light is not blocked by the specimen, thereby enabling automatic observation.

  In the optical microscope of the present invention, it is preferable that the light source is an array including a plurality of light sources.

  A mechanism for physically rotating the light source is not necessary, and the shape of the light beam can be arbitrarily selected by appropriately selecting an array to be lit. Further, by controlling the array to be lit, the light beam can be rotated around the optical axis.

  In the optical microscope of the present invention, preferably, the specimen held on the stage is a well plate or a dish-like culture container capable of holding a culture solution.

  The optical microscope of the present invention is useful when observing a specimen made of cultured cells together with a culture solution on a stage, and in particular, the rotational position of the light source is controlled in correspondence with a culture vessel such as a well plate or a dish. Therefore, appropriate oblique illumination can be performed without obstructing oblique illumination by the shape of the specimen.

  In the optical microscope of the present invention, preferably, a transmission illumination optical system is applied to phase difference observation.

  When the phase difference objective lens is used, the illumination optical system of the present invention can be applied to the phase difference observation as it is for the illumination optical system.

Next, the basic configuration of the optical microscope of the present invention will be described with reference to FIG.
The optical microscope of the present invention enlarges a light source 1 of a halogen lamp, a transmission illumination optical system 4 that guides light from the light source 1 to a specimen 3 held by a stage 2, and an image of an object to be observed in the specimen 3. And an imaging optical system 5 for this purpose.

  The transmission illumination optical system 4 includes condenser lenses 41 and 42, a light shielding unit 43 that asymmetrically shields and illuminates a light beam from the light source 1, a condenser lens 44, and a light diffusion plate 45. The light blocking means 43 includes, for example, a disk-shaped blocking plate 432 having a fan-shaped hole 431 in the optical axis direction, and a motor 433 and a gear mechanism that rotate the blocking plate 432 around the optical axis according to a command from the computer 6 434 and a rotation holding mechanism (not shown).

  The imaging optical system 5 includes a phase difference objective lens 52 that is directed to the bottom surface of the specimen 3 and has a phase film 51, and an imaging lens and an imaging device (not shown). The phase film 51 is set at the pupil position of the phase difference objective lens 52, and a partial fan-shaped hole 431 of the blocking plate 432 is projected onto the phase film 51 of the phase difference objective lens 52.

  The stage 2 holding the specimen 3 can be moved in a direction perpendicular to the optical axis by an XY drive mechanism (not shown). The computer 6 controls the XY drive mechanism and the light shielding means 43 in conjunction with each other. In some cases, a rotation mechanism that rotates the stage 2 around the optical axis may be provided together with the XY drive mechanism. In addition to the XY drive mechanism, only the stage 2 may be rotated. Alternatively, only the stage 2 may be directly rotated without providing the XY drive mechanism.

Next, the optical microscope of Example 1 described above will be described in more detail together with the control / observation method.
Light emitted from the light source 1 of the halogen lamp is projected onto the blocking plate 432 by the condenser lenses 42 and 43. For example, a light diffusing plate 45 is inserted in the middle of the optical path so that the halogen lamp image is projected onto the blocking plate 432 uniformly.

  As shown in FIG. 2, the blocking plate 432 is partially provided with a fan-shaped hole 431. The blocking plate 432 is driven and controlled by a command from the computer 6 by a motor 433, a gear mechanism 434, and a rotation holding mechanism (not shown), and is configured to rotate about the optical axis. Is controlled by the computer 6 based on the XY coordinate position information. That is, the XY coordinate position information of the stage 2 is sequentially acquired by the computer 6, and the rotational position of the blocking plate 432 is sequentially controlled by the computer 6 based on the XY coordinate position information. The position information of the stage 2 is not based on the coordinate position of the XY drive mechanism, and the position information of the stage 2 may be obtained by other methods.

Next, the control of the rotational position of the blocking plate 432 based on the XY coordinate position information of the stage 2 acquired by the XY drive mechanism will be described in detail.
Of the luminous flux emitted from the light source 1, the luminous flux that has passed through the fan-shaped hole 431 of the blocking plate 432 passes through the condenser lens 44, passes through the culture vessel 31 containing the culture solution, and irradiates cells (not shown), A magnified image is obtained by the objective lens 52 and an imaging lens (not shown), and is captured by a camera (not shown). The culture vessel 31 is mounted on a stage 2 having an XY drive mechanism (not shown), and the XY position is moved one after another according to a command from the computer 6 so that almost the entire area is imaged, and images are sequentially captured.

  Then, when the culture vessel 31 is moved by the XY drive mechanism and sequentially imaged in accordance with a command from the computer 6, it passes through the fan-shaped hole 431 of the blocking plate 432 according to the XY coordinate position information of the stage 2 by the computer 6 at that time. The rotational position of the blocking plate 432 is controlled by the computer 6 so that light is not blocked by the wall of the culture vessel 31.

  In other words, the rotational position of the shielding plate 432 is determined by the light passing through the fan-shaped hole 431 of the shielding plate 432 on the wall of the culture vessel 31 according to the XY coordinate position information of the stage 2 stored in advance or sequentially acquired by the computer 6. It is controlled so that it is not obstructed. Alternatively, the rotational position of the blocking plate 432 is automatically determined by a preset calculation formula in relation to the culture container 31 used and the XY coordinate position of the stage 2. This calculation formula is appropriately selected according to the type of microplate or dish used as the culture vessel 31.

  Here, the relationship between the imaging position and the rotational position of the blocking plate 432 will be described in more detail with reference to FIGS. 3A is a plan view of the culture vessel 31, and FIG. 3B is a front view. In FIG. 3A, a, b, c, and d indicate positions where imaging is performed. When imaging the position a from 9 o'clock to 12 o'clock as viewed from the center of the culture vessel 432, the blocking plate 432 has a fan-shaped hole 431 with respect to the optical axis from 3 o'clock to 6 o'clock as shown in FIG. The position is determined by a calculation formula so as to be the hour position, and the motor 433 is rotated by the computer 6 so that the blocking plate 432 is controlled to a predetermined position. Similarly, when imaging b, the blocking plate 432 is rotated to the position of FIG. 4B, and when imaging c, the blocking plate 432 is rotated to the position of FIG. Rotates the blocking plate 432 to the position shown in FIG.

  In order to rotate the stage 2 on which the culture vessel 31 is placed relative to the blocking plate 432 about the optical axis, the stage 2 on which the culture vessel 31 is placed can be cut off even if the stage 2 on which the culture vessel 31 is placed is rotated. The plate 432 may be rotated, or both the stage 2 and the blocking plate 432 may be rotated. In any case, oblique illumination may be performed from the direction in which the light passing through the fan-shaped hole 431 of the blocking plate 432 is not blocked by the wall of the culture vessel 31.

In this embodiment, the objective lens is the phase difference objective lens 52, and the phase film 51 is set at the pupil position, so that the partial fan-shaped hole 431 of the blocking plate 432 is projected onto the phase film 51. It has become. Thereby, phase difference observation can be performed.
Note that the objective lens does not necessarily need to have a phase film, and when there is no phase film, the objective lens functions as normal transmission oblique illumination.

  Further, in the present embodiment, an example of a petri dish is illustrated as the culture container 31, but a microplate 33 having a large number of wells 32 may be used as shown in FIG. In this case, each of the wells 32 is considered as a dish, and the blocking plate 432 is rotated for each well 32 in accordance with, for example, the imaging positions a, b, c, and d.

  Further, when the objective lens is not a phase difference objective lens, the opening of the blocking plate 432 does not need to be a fan-shaped hole 431, and the opening of the blocking plate 432 is a rectangular hole 435 as shown in FIG. There may be.

In addition, when a shield plate 432 having a fan-shaped hole 431 as shown in FIG. 2 is used, it is necessary to replace the shield plate with a different opening width in order to adjust the amount of declination. is there. Further, when the oblique illumination is not required, an operation for removing the blocking plate occurs.
In order to solve these problems, a slider 8 as shown in FIG. 7 may be used instead of the disc-shaped blocking plate 432. The slider 8 has a substantially fingertip-shaped hole 81 at the center in the optical axis direction, and can slide about the optical axis while sliding in the left-right direction in FIG. 7A.

  When the oblique illumination is not used, uniform illumination can be performed without blocking the light flux as shown in FIGS. Further, as shown in FIGS. 7C and 7D, by moving the slider 8 perpendicular to the optical axis, it is possible to adjust the amount of blocking the light beam 82 and to adjust the contrast. is there.

  3A, the contrast position can be freely adjusted by rotating the slider 8 with respect to the optical axis as shown in FIG. However, oblique illumination can be performed without blocking light on the wall of the culture vessel 31. Specifically, when imaging a, the slider 8 is rotated to the position shown in FIG. 8A, and when imaging b, the slider 8 is rotated to the position shown in FIG. Rotates the slider 8 to the position shown in FIG. 6C, and rotates the slider 8 to the position shown in FIG.

  In this embodiment, in place of the light source 1 and the light shielding means 43, the light source 1 in which an array of a plurality of LEDs 7 as shown in FIG. The array of the plurality of LEDs 7 may be arranged so as to go around the concentric circle as shown in FIG. 9B, or may be arranged only in a part of the concentric circle as shown in FIG. Good.

9B, when the array of LEDs 7 is arranged so as to go around the concentric circle, the LED 7 to be turned on among the LEDs 7 provided on the concentric circle by the XY coordinate position information of the stage 2 by the computer 6. Select and light up.
For example, in FIG. 9B, the LED 71 between 2 o'clock and 4 o'clock is lit, and the other LEDs 72 are not lit. By selecting and changing the LED 7 to be lit, the luminous flux can be made asymmetric with respect to the optical axis. Further, by switching so that the LEDs 7 to be lit are sequentially rotated, the light beam can be relatively rotated around the optical axis.

  For example, when imaging the positions a, b, c, and d in FIG. 3A, the contrast position is set by sequentially rotating the LEDs 7 that are turned on with respect to the optical axis as shown in FIG. While adjusting freely, oblique illumination can be performed without blocking light on the wall of the culture vessel 31. Specifically, when imaging a, the LED 71 at the position shown in FIG. 10A is turned on. When imaging b, the LED 71 at the position shown in FIG. The LED 71 at the position shown in FIG. (C) is turned on, and the LED 71 at the position shown in FIG.

9C, when the array of LEDs 7 is arranged only in a part of the concentric circle, the computer 6 rotates the light source 1 by the motor 433 based on the XY coordinate position information of the stage 2.
For example, in FIG. 9C, the light source 1 is rotated so that the LED 7 between 2 o'clock and 4 o'clock is positioned. By changing the rotational position of the light source 1, the light beam can be rotated about the optical axis.

  The present invention is not limited to an inverted optical microscope equipped with a phase difference objective lens, and functions as transmission oblique illumination when observing a specimen in which cells and a culture solution are placed in a transparent container with an optical microscope. It can be widely applied not only to observation.

DESCRIPTION OF SYMBOLS 1 Light source 2 Stage 3 Sample 31 Culture container 32 Well 33 Microplate 4 Transmission illumination optical system 41 Condensing lens 42 Condensing lens 43 Light-shielding means 431 Fan-shaped hole 432 Blocking plate 433 Motor 434 Gear mechanism 435 Rectangular hole 44 Capacitor Lens 45 Light diffusing plate 5 Imaging optical system 51 Phase film 52 Phase difference objective lens 6 Computer 7 LED
71 Lit LED
72 Unlit LED
8 Slider 81 Substantially fingertip-shaped hole 82

Claims (8)

  An optical microscope comprising a stage for holding a specimen and a transmission illumination optical system for illuminating the specimen, wherein the transmission illumination optical system includes light shielding means for asymmetrically shielding and illuminating a light beam from a light source. And an optical microscope characterized in that the light shielding means is configured to rotate relative to the stage about an optical axis.   The optical microscope according to claim 1, wherein the rotational position of the light shielding unit is automatically controlled in accordance with the sample based on the position information of the stage.   The optical microscope according to claim 1, wherein the light shielding unit is configured such that a shape of a light beam that has passed through the light shielding unit is a fan shape.   An optical microscope comprising a stage for holding a specimen and a transmission illumination optical system for illuminating the specimen, wherein the transmission illumination optical system is provided with a light source that can be turned on so that a light beam is asymmetric with respect to the optical axis And the light source is configured such that the light beam rotates relative to the optical axis or the lighting position is selected.   The optical microscope according to claim 4, wherein a rotation position or a lighting position of the light source is automatically controlled in accordance with the specimen based on the position information of the stage.   The optical microscope according to claim 4 or 5, wherein the light source is configured by an array of a plurality of light sources.   The optical microscope according to claim 1, wherein the specimen held on the stage is a well plate or a dish-like culture container capable of holding a culture solution.   The optical microscope according to claim 1, wherein the transmission illumination optical system is applied to phase difference observation.

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