JP2010191170A - Three-dimensional microscope, and observation and measuring method using three dimensional microscope - Google Patents
Three-dimensional microscope, and observation and measuring method using three dimensional microscope Download PDFInfo
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Abstract
Description
本発明は、主として観察対象物の大きさが数μm程度の微小体を立体的に顕微鏡観察・測定するに際し、有用な三次元顕微鏡装置及び同装置を用いた観察・測定法に関する。 The present invention mainly relates to a useful three-dimensional microscope apparatus and an observation / measurement method using the apparatus when three-dimensionally observing and measuring a microscopic object having an observation object size of about several μm.
三次元情報が取得可能な従来の顕微鏡装置としては、代表例として、以下に述べる2つの装置がある。 As a typical example of a conventional microscope apparatus capable of acquiring three-dimensional information, there are two apparatuses described below.
第1は、追跡型顕微鏡といわれるもので、観察対象物が焦点面に入るように自動で同対象物の位置を動かすようにする装置である。 The first is a tracking microscope, and is a device that automatically moves the position of the object so that the object is in the focal plane.
しかし、この装置はその原理上奥行き方向の解像度が同装置のレンズ系の焦点深度の程度しかないという課題、また、対象物の大きさが数μmというような微小の物体であるとその輪郭が検出できず十分な観察・測定が不可能という課題がある。 However, this device has a problem that the resolution in the depth direction is only about the depth of focus of the lens system of the device, and the contour of the object is a minute object with a size of several μm. There is a problem that it cannot be detected and sufficient observation and measurement are impossible.
さらにまた、対象物の回転等の姿勢に関する情報が得られないという課題もある。 Furthermore, there is a problem that information on the posture such as rotation of the object cannot be obtained.
第2は、全焦点型顕微鏡といわれるもので、対物レンズを高速で走査し、対象物が焦点面に入った部分のみを検出し、三次元像を構築する装置である。 The second is an all-focus microscope, which is an apparatus that constructs a three-dimensional image by scanning an objective lens at a high speed and detecting only a portion where an object enters the focal plane.
しかしこの装置も又、奥行き方向の解像度が同装置のレンズ系の焦点深度の程度しかないという課題、また、対象物の大きさが数μmというような微小の物体であるとその輪郭が検出できず十分な観察・測定が不可能という課題がある。 However, this device also has the problem that the resolution in the depth direction is only the depth of focus of the lens system of the device, and the contour of the object can be detected if it is a minute object such as several μm in size. Therefore, there is a problem that sufficient observation and measurement are impossible.
さらにまた、対象物の回転等の姿勢に関する情報が広範囲に得られないという課題や対物レンズの走査方向の範囲、つまり可測定範囲が制限されるという課題もある。 Furthermore, there is a problem that information on the posture such as the rotation of the object cannot be obtained in a wide range and a range in the scanning direction of the objective lens, that is, a measurable range is limited.
本発明は、前述の第1、第2の顕微鏡装置の課題である、対象物の回転等の姿勢に関する情報が得られない、又は広範囲に得られないという問題を解決しようとするものである。 The present invention seeks to solve the problem of the above-described first and second microscope apparatuses, in which information relating to the posture of the object such as rotation cannot be obtained or cannot be obtained in a wide range.
微小物体であるバクテリアの多くは、らせん形のべん毛を回転することで溶液中を直進遊泳するが、壁面や液面などの境界近傍においては、べん毛の回転に伴う周囲の溶液の流れが境界と干渉することにより、バクテリアの遊泳軌跡は壁面に沿った円弧を描く。このような運動を観察・測定するためには、境界に対するバクテリアの三次元位置や回転等の姿勢に関する情報が必要となるが、本発明は、これら情報の取得が広範囲にわたって可能
となる。
Many bacteria, which are microscopic objects, move straight in the solution by rotating the spiral flagella. However, in the vicinity of the boundary such as the wall surface or liquid surface, the flow of the surrounding solution accompanying the rotation of the flagella By interfering with the boundary, the bacterial trajectory draws an arc along the wall. In order to observe and measure such movement, information on the three-dimensional position of bacteria and the posture such as rotation with respect to the boundary is required, but the present invention can acquire such information over a wide range.
本発明請求項1に係る発明は、観察対象物の画像情報を得るための光源、対物レンズ、撮像体よりなる第一の撮像装置と、前記第一の撮像装置とは異なる方向より前記観察対象物の画像情報を得るための光源、対物レンズ、撮像体よりなる第二の撮像装置とよりなり、前記第一の撮像装置より得られた第一の画像情報と前記第二の撮像装置より得られた第二の画像情報より、前記観察対象物の三次元情報を得るようにするとともに、前記観察対象物の特定点の位置情報を複数時点において取得し、それら取得された位置情報より前記観察対象物の回転等の姿勢に関する情報を得ることを特徴とする三次元顕微鏡装置である。 According to the first aspect of the present invention, there is provided a first imaging device including a light source, an objective lens, and an imaging body for obtaining image information of an observation target, and the observation target from a direction different from the first imaging device. A second imaging device comprising a light source, an objective lens, and an imaging body for obtaining image information of an object, and obtained from the first image information obtained from the first imaging device and the second imaging device. 3D information of the observation object is obtained from the obtained second image information, and position information of specific points of the observation object is obtained at a plurality of time points, and the observation is obtained from the obtained position information. It is a three-dimensional microscope apparatus characterized by obtaining information related to a posture such as rotation of an object.
本発明請求項2に係る発明は、観察対象物の画像情報を得るための光源、対物レンズ、撮像体よりなる第一の撮像装置と、前記第一の撮像装置とは異なる方向より前記観察対象物の画像情報を得るための光源、対物レンズ、撮像体よりなる第二の撮像装置とよりなり、前記第一の撮像装置より得られた第一の画像情報と前記第二の撮像装置より得られた第二の画像情報より、前記観察対象物の三次元情報を得るようにした装置において、2枚の板体間に留保された液中に前記観察対象物を収容するようになすとともに、前記2枚の板体間の間隔を、一方の対物レンズの視野に入らない範囲で、かつ当該2枚の板体間の液が表面張力により留保されるように設定することを特徴とする三次元顕微鏡装置である。 According to a second aspect of the present invention, there is provided a first imaging device including a light source, an objective lens, and an imaging body for obtaining image information of an observation target, and the observation target from a direction different from the first imaging device. A second imaging device comprising a light source, an objective lens, and an imaging body for obtaining image information of an object, and obtained from the first image information obtained from the first imaging device and the second imaging device. From the obtained second image information, in the apparatus for obtaining the three-dimensional information of the observation object, the observation object is accommodated in the liquid retained between the two plates, The tertiary is characterized in that the interval between the two plates is set in a range that does not enter the field of view of one objective lens, and the liquid between the two plates is retained by surface tension. The original microscope device.
本発明請求項3に係る発明は、観察対象物の画像情報を得るための光源、対物レンズ、撮像体よりなる第一の撮像装置と、前記第一の撮像装置とは異なる方向より前記観察対象物の画像情報を得るための光源、対物レンズ、撮像体よりなる第二の撮像装置とよりなり、前記第一の撮像装置より得られた第一の画像情報と前記第二の撮像装置より得られた第二の画像情報より、前記観察対象物の三次元情報を得るようにした装置を用いて、前記観察対象物の特定二点の位置情報から同特定二点間の距離を複数個取得して同特定二点間の距離の平均値を算出するとともに、前記観察対象物の特定二点の距離を前記平均値となるように位置情報の補正値を求め前記位置情報の最尤値を求めることを特徴とする三次元顕微鏡装置を用いた観察・測定法である。 According to a third aspect of the present invention, there is provided a first imaging device including a light source, an objective lens, and an imaging body for obtaining image information of an observation target, and the observation target from a direction different from the first imaging device. A second imaging device comprising a light source, an objective lens, and an imaging body for obtaining image information of an object, and obtained from the first image information obtained from the first imaging device and the second imaging device. Using a device that obtains three-dimensional information of the observation object from the obtained second image information, a plurality of distances between the two specified points are obtained from position information of the two specific points of the observation object And calculating an average value of the distance between the two specified points, obtaining a correction value of the position information so that the distance between the two specified points of the observation object becomes the average value, and calculating the maximum likelihood value of the position information. Observation and measurement using a 3D microscope device A.
本発明は、前述のような構成からなるので、従来の装置では不可能であった微小体の回転等の姿勢に関する情報が広範囲に得られることとなり、バクテリア等の境界に対する挙動の観察・測定に多大な効果を奏する。
即ち、本発明は、対物レンズ系の焦点深度の数倍の範囲において、観察・測定可能であり、解像度が焦点深度に依存しないという効果を奏する。
Since the present invention is configured as described above, information on postures such as rotation of microscopic bodies, which was impossible with conventional devices, can be obtained over a wide range, and observation and measurement of behavior with respect to boundaries of bacteria and the like can be obtained. There is a great effect.
That is, the present invention can observe and measure in the range of several times the focal depth of the objective lens system, and has an effect that the resolution does not depend on the focal depth.
本発明の実施例につき、以下、図面を用いて説明する。図1は本発明の実施例の概略的
構成を示している。図1において右下に示す方向にx、y、z軸をとる。1は光源としての透過光源、2は位相差コンデンサ、3は対物レンズ、4は対物レンズ位置調節ハンドル、5は撮像体の一例としてのCCDカメラで、13は光路折り曲げミラーであり、1〜5、13で1つの顕微鏡ユニット、即ち第一の撮像装置の一例を構成している。13の光路折り曲げミラーは、対物レンズを通った光をCCDカメラ5に入れるために設けた。
Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 shows a schematic configuration of an embodiment of the present invention. In FIG. 1, the x, y, and z axes are taken in the direction shown at the lower right. 1 is a transmissive light source as a light source, 2 is a phase difference condenser, 3 is an objective lens, 4 is an objective lens position adjustment handle, 5 is a CCD camera as an example of an image pickup body, 13 is an optical path bending mirror, 1-5 , 13 constitutes one microscope unit, that is, an example of the first imaging device. Thirteen optical path bending mirrors were provided to allow the light passing through the objective lens to enter the CCD camera 5.
また、もう1つの顕微鏡ユニット、即ち第二の撮像装置の一例は、透過光源6、位相差コンデンサ7、対物レンズ8、xyz調節台9、撮像体の一例としてのCCDカメラ10、ビデオマイクロスコープ14から構成される。 Another example of the microscope unit, that is, the second imaging device, includes a transmission light source 6, a phase difference capacitor 7, an objective lens 8, an xyz adjustment table 9, a CCD camera 10 as an example of an imaging body, and a video microscope 14. Consists of
1〜5、13からなる顕微鏡ユニットは、既製の顕微鏡(オリンパス社、IX−71)を用いているが、6〜10、14から構成される顕微鏡ユニットはそれらを設置する顕微鏡本体が無いため、光軸合わせを行う必要があり、また対物レンズ8を通った光がCCDカメラ10において像を結ぶためのビデオマイクロスコープ14が必要となる。 The microscope unit consisting of 1 to 5 and 13 uses an off-the-shelf microscope (Olympus, IX-71), but the microscope unit composed of 6 to 10 and 14 has no microscope main body for installing them, It is necessary to align the optical axes, and a video microscope 14 is required for the light passing through the objective lens 8 to form an image in the CCD camera 10.
カメラ5、10に結ばれた像は図示しない画像取り込みボードによりハードディスクに同期取込みする。観察対象物に応じて透過光源1、6を落射光源にして、位相差コンデンサ2、7を無くしても良い。光源の光は可視光に限定されるわけではなく、赤外線、紫外線やX線でもよい。上記2つの撮像装置における光軸は、異なる方向、つまり平行でないことが要求される。本実施例においては、2つの光軸が直交するようにした。 Images connected to the cameras 5 and 10 are synchronously captured in the hard disk by an image capturing board (not shown). Depending on the observation object, the transmission light sources 1 and 6 may be incident light sources, and the phase difference capacitors 2 and 7 may be eliminated. The light of the light source is not limited to visible light, but may be infrared, ultraviolet or X-ray. The optical axes in the two imaging devices are required to be different directions, that is, not parallel. In this embodiment, the two optical axes are orthogonal to each other.
上記2つの顕微鏡ユニットにおける光軸が垂直に交わるようにするため、1〜5、13から構成される顕微鏡ユニットを組み立て、その光軸にできるだけ垂直な光軸を持つように6〜10、14を配置する。その後、xyz調節台9を用いて2つの光軸が交わるようにし、xyz調節台9をx方向に調節することで、2つの対物レンズ3、8の焦点面の交線が2つのCCDカメラ5、10いずれの視野にも入るようにする。2つの光軸は厳密に交わる必要はなく、CCDカメラ5、10どちらの視野にも同一の観察対象物が結像されていればよい。また2つの光軸が垂直であることも、後の解析に便利になるためであり、本発明の原理においては厳密に垂直であることを要求しない。 In order to make the optical axes of the two microscope units perpendicular to each other, a microscope unit composed of 1 to 5 and 13 is assembled, and 6 to 10 and 14 are arranged so as to have an optical axis as perpendicular to the optical axis as possible. Deploy. Thereafter, the two optical axes intersect with each other using the xyz adjusting table 9 and the xyz adjusting table 9 is adjusted in the x direction so that the line of intersection of the focal planes of the two objective lenses 3 and 8 is two CCD cameras 5. 10 to be in any field of view. The two optical axes do not need to cross each other exactly, and it is only necessary that the same observation object is imaged in the fields of view of the CCD cameras 5 and 10. Also, the fact that the two optical axes are perpendicular is convenient for later analysis, and the principle of the present invention does not require that they be strictly perpendicular.
2つの対物レンズ3、8はお互いにぶつからない配置をとる必要がある。高倍率の対物レンズを用いる場合は作動距離が短いため、通常の20倍以上の対物レンズを用いてその光軸が直交し、かつ2つの焦点面の交線がいずれものCCDカメラの視野に入ることは困難である。これを防ぐため、作動距離の長い対物レンズを用いる必要がある。本実施例のように、2つの対物レンズの光軸が垂直な場合、一方の対物レンズの作動距離は他方の対物レンズの筒の最大半径、つまり対物レンズの最外縁径の半分の長さよりも長いことが必要である。 The two objective lenses 3 and 8 need to be arranged so as not to collide with each other. When a high-magnification objective lens is used, the working distance is short, so the optical axis is orthogonal using a normal objective lens of 20 times or more, and the intersection of the two focal planes enters the field of view of any CCD camera. It is difficult. In order to prevent this, it is necessary to use an objective lens having a long working distance. As in this embodiment, when the optical axes of two objective lenses are vertical, the working distance of one objective lens is larger than the maximum radius of the cylinder of the other objective lens, that is, the length of half the outermost edge diameter of the objective lens. It needs to be long.
また、観察対象物を固定するためのサンプルホルダ11に上記2つの対物レンズがぶつからないように工夫する必要がある。本実施例においては既製の顕微鏡のステージを取り除き、代わりに金属板を用いて小型のステージを作成し、12に示すxy調節台に固定した棒にステージを吊り下げることで、従来のステージと同様にxy方向に移動可能でかつ、上記2つの対物レンズがぶつからないサンプルホルダを作成した。 Further, it is necessary to devise so that the two objective lenses do not collide with the sample holder 11 for fixing the observation object. In this embodiment, the stage of the ready-made microscope is removed, a small stage is created using a metal plate instead, and the stage is suspended from a rod fixed to the xy adjustment base shown in FIG. A sample holder that can move in the xy direction and does not collide with the two objective lenses was prepared.
図1に示す装置を用いてゾウリムシを観察したときの画像を図2に示す。図2(a)(b)は、図1においてそれぞれCCDカメラ5、10の画像であり、2つの対物レンズ3、8の光軸が垂直な場合、図2に示すx、y、z方向はそれぞれ図1のx、y、z方向と対応する。2つの光軸が垂直でない場合には、垂直からの傾き角をあらかじめ測っておく必要がある。図2(a)(b)ともに、画面左側に見えるのがゾウリムシ、右側に見える黒いものは溶液中のゴミであり、2方向から同一物体を観察できることがわかる。ここで
ゾウリムシの片方の端を座標原点とする。つまり、図2(a)におけるゾウリムシの右端をx、y座標の原点、図2(b)におけるゾウリムシの右端をx、zの原点とする。
FIG. 2 shows an image obtained when the Paramecium is observed using the apparatus shown in FIG. 2A and 2B are images of the CCD cameras 5 and 10 in FIG. 1, respectively. When the optical axes of the two objective lenses 3 and 8 are vertical, the x, y, and z directions shown in FIG. These correspond to the x, y, and z directions in FIG. When the two optical axes are not vertical, it is necessary to measure the inclination angle from the vertical in advance. 2 (a) and 2 (b), it can be seen that Paramecium is seen on the left side of the screen, and black thing seen on the right side is dust in the solution, and the same object can be observed from two directions. Here, one end of the Paramecium is the coordinate origin. That is, the right end of Paramecium in FIG. 2A is the origin of x and y coordinates, and the right end of Paramecium in FIG. 2B is the origin of x and z.
そうすると、あらかじめ2つの画像の縮尺を測定しておけば、ゾウリムシの原点でないほうの端について、位置情報である位置座標が取得可能である。ゾウリムシのような細長い物体についてその両端の位置座標を各時刻において取得できれば、三次元の並進および回転運動についての情報が得られる。 Then, if the scales of the two images are measured in advance, the position coordinates as the position information can be acquired for the end that is not the origin of the Paramecium. If the position coordinates of both ends of a long and narrow object such as Paramecium can be obtained at each time, information about three-dimensional translation and rotational motion can be obtained.
従来の三次元顕微鏡観察手法においては、観察対象物が対物レンズの焦点面から外れたときの輪郭のボケ具合を検出して視野内における奥行き情報を得ていたため、奥行き方向の解像度が用いた対物レンズの焦点深度の程度しかなかったが、本発明の手法ではxyz全ての方向について直接観察するので、xyzどの方向においても解像度は高く、原理的には光の波長程度の解像度を有する。 In the conventional three-dimensional microscope observation method, the depth information in the field of view is obtained by detecting the degree of blurring of the contour when the observation object deviates from the focal plane of the objective lens. Although there was only the depth of focus of the lens, since the method of the present invention directly observes in all directions of xyz, the resolution is high in any direction of xyz, and in principle, has a resolution of the wavelength of light.
ゾウリムシのように観察対象物が運動する場合、従来の手法である追跡型顕微鏡を用いる場合はピントが外れることなく観察できるが、本発明による手法の場合は観察対象物の完全な追跡は不可能であり、鮮明な画像を得ることができないため、取得した座標には誤差を含む。例えば図2(a)におけるゾウリムシの左端のy座標と図2(b)におけるゾウリムシの左端のy座標は本来等しいはずであるが、像が不鮮明になるにつれてその差は大きくなる。 When the observation object moves like a Paramecium, it can be observed without being out of focus when using the conventional tracking microscope, but the method of the present invention cannot completely track the observation object. Since a clear image cannot be obtained, the acquired coordinates include an error. For example, the y coordinate of the left edge of a Paramecium in FIG. 2A and the y coordinate of the left edge of a Paramecium in FIG. 2B should be essentially the same, but the difference increases as the image becomes unclear.
このような場合に、求める位置座標の最尤値を求めるための補正が必要となる。まず、観察対象物はゾウリムシのように細長く、両端が存在するものとする。各時刻における観察対象物の両端の位置座標から、両端間の長さLを求める。各時刻におけるLは測定が精密ならば一定の値のはずであるが、ピントのズレなどにより異なった値をとる。 In such a case, correction for obtaining the maximum likelihood value of the position coordinates to be obtained is necessary. First, it is assumed that the observation object is elongated like a Paramecium and has both ends. From the position coordinates of both ends of the observation object at each time, a length L between both ends is obtained. L at each time should be a constant value if the measurement is precise, but takes a different value due to a focus shift or the like.
そこで、各時刻におけるLの平均Laveを求め、測定した両端間の距離が常にLaveとなるように、測定した座標の補正値を求める。 Therefore, an average L of L at each time is obtained, and a correction value of the measured coordinates is obtained so that the measured distance between both ends is always Love.
さらに、補正した座標と初めに測定した座標との差が出来るだけ少なくなるように、ラグランジュの未定乗数法を用いて補正値を求める。これにより、ピントのずれによる誤差の影響を可能な限り除去した位置座標の最尤値が求まる。撮像装置を3つ以上としても良く、その場合補正の精度は向上する。 Further, a correction value is obtained by using the Lagrange's undetermined multiplier method so that the difference between the corrected coordinate and the initially measured coordinate becomes as small as possible. Thereby, the maximum likelihood value of the position coordinate from which the influence of the error due to the focus shift is removed as much as possible is obtained. Three or more imaging devices may be provided, and in this case, the accuracy of correction is improved.
三次元観察が有効であるのは、多くの場合、観察対象物が壁面や水面などの界面近傍を運動するときであるが、本発明による手法を用いて境界近傍を観察する際には、境界面に平行に対物レンズの光軸が存在する場合に境界面からの反射光が対物レンズに入り、境界近傍を運動する対象物のコントラストが得られないという問題点がある。この問題を解決するために、ガラスのような透明板体を用いて微小な試料容器を作る方法が挙げられる。 In many cases, the three-dimensional observation is effective when the observation object moves in the vicinity of the interface such as the wall surface or the water surface, but when observing the vicinity of the boundary using the method according to the present invention, the boundary is observed. When the optical axis of the objective lens exists parallel to the surface, there is a problem that the reflected light from the boundary surface enters the objective lens and the contrast of the object moving near the boundary cannot be obtained. In order to solve this problem, there is a method of making a minute sample container using a transparent plate such as glass.
本実施例においては、図3に示すように、ガラスで囲まれた空間内に試料溶液を注入する。このとき、溶液の表面張力により、周囲を密封することなく溶液を留保することができる。この空間のサイズは、観察対象物の大きさにより自由に変えても構わないが、表面張力により液滴が動くことなく留保される必要がある。試料溶液20を2枚のガラス16、17で挟んだものをサンプルホルダ15に立てて固定し、2つの方向から対物レンズ18、19を用いて観察する。 In this embodiment, as shown in FIG. 3, the sample solution is injected into a space surrounded by glass. At this time, the solution can be retained without sealing the periphery due to the surface tension of the solution. The size of this space may be freely changed depending on the size of the object to be observed, but it is necessary to retain the droplet without moving due to the surface tension. A sample solution 20 sandwiched between two glasses 16 and 17 is fixed on a sample holder 15 and observed using objective lenses 18 and 19 from two directions.
図3に示すxyz軸の方向は、図1に示すものと対応している。図3における試料溶液20の下面にある自由表面近傍を観察領域とすると、この自由表面の面積が狭いほど対物レンズ19に入る境界からの反射光は少なくなる。ただし、ガラス16、17の間隔を狭
くしすぎると、対物レンズ18の視野内にガラス16、17が入ってしまい、対物レンズ18から得られる像が不鮮明になってしまう。
The direction of the xyz axis shown in FIG. 3 corresponds to that shown in FIG. If the vicinity of the free surface on the lower surface of the sample solution 20 in FIG. 3 is an observation region, the smaller the area of the free surface, the less the reflected light from the boundary entering the objective lens 19. However, if the distance between the glasses 16 and 17 is too small, the glasses 16 and 17 enter the field of view of the objective lens 18 and the image obtained from the objective lens 18 becomes unclear.
このため、ガラス16、17間の距離は、対物レンズ18の視野に入らない程度に広くかつ、出来るだけ狭いことが望ましい。また、この自由表面に薄いガラスなどでふたをすることにより、固体表面近傍における三次元観察も可能になるものと考えられる。境界からの反射を防ぐ別の方法として、どちらの対物レンズにも境界からの反射光が入らないようにサンプルを保持する角度を工夫するという手法も考えられる。 For this reason, it is desirable that the distance between the glasses 16 and 17 be as wide as possible and not as narrow as possible without entering the field of view of the objective lens 18. Further, it is considered that three-dimensional observation in the vicinity of the solid surface can be performed by covering the free surface with thin glass or the like. As another method for preventing reflection from the boundary, a method of contriving an angle for holding the sample so that reflected light from the boundary does not enter either objective lens is also conceivable.
図2におけるゾウリムシの画像も、上記の方法で試料作成をしており、図2(b)の下部の黒い部分が自由表面であるが、境界からの反射光は少なく、鮮明な像が得られている。 The image of Paramecium in FIG. 2 is also prepared by the above method, and the black part in the lower part of FIG. 2B is a free surface, but there is little reflected light from the boundary and a clear image is obtained. ing.
本実施例の手法は、顕微鏡の形状の制限があったため、上記のような形をとったが、境界からの反射光が対物レンズに入らないような形態であれば何でもよい。例えば、透明板体を透さない方向から観察できるのであれば、試料を留保する容器は透明でなくても良いし、微小管から垂れている微小な液滴や、単にガラス板上の微小液滴を複数方向から観察する、という方法でも良い。 The method of the present embodiment has the above-mentioned shape because of the limitation of the shape of the microscope, but may be anything as long as the reflected light from the boundary does not enter the objective lens. For example, if observation is possible from a direction that does not pass through a transparent plate, the container for retaining the sample does not have to be transparent, a minute droplet hanging from a microtube, or a simple liquid on a glass plate. A method of observing the droplet from a plurality of directions may be used.
本発明の方法を用いて、ビブリオ菌の自由表面近傍における遊泳運動を観察した。ビブリオ菌の大きさは数マイクロメートルであるため、高倍率の対物レンズが必要であるが、対物レンズどうしがぶつからないように、対物レンズ3にはオリンパス社のSLMPLN50×(作動距離18ミリ)を用いた。 Using the method of the present invention, the swimming movement near the free surface of Vibrio was observed. Since the size of Vibrio is several micrometers, a high-magnification objective lens is required. However, in order to prevent the objective lenses from colliding with each other, Olympus SLMPLN50 × (working distance of 18 mm) is used for the objective lens 3. Using.
図4において、(a)は図1におけるCCDカメラ5の映像より得たxy平面上の菌の遊泳軌跡、(b)は図1におけるCCDカメラ10のより得たxz平面上の菌の遊泳軌跡であり、(c)は(a)(b)2つの画像を合成して得た菌の三次元軌跡を表す。映像のフレームごとに菌体の先頭位置の座標を取得し、これらをグラフにプロットすることで、細菌の遊泳軌跡を表した。 4, (a) is a fungus swimming locus on the xy plane obtained from the image of the CCD camera 5 in FIG. 1, and (b) is a fungus swimming locus on the xz plane obtained from the CCD camera 10 in FIG. (C) represents a three-dimensional trajectory of the fungus obtained by synthesizing two images (a) and (b). The coordinates of the start position of the bacterial cells were obtained for each frame of the video, and these were plotted on a graph to represent the bacterial trajectory.
図5において、(a)は菌体の角度を定義するための図、(b)は角度θと自由表面からの距離dの時間変化、(c)は角度φと自由表面からの距離dの時間変化を示している。ビブリオ菌のサイズは数マイクロメートルと小さいが、図4、5から三次元位置と回転に関する情報が得られることがわかる。 5, (a) is a diagram for defining the angle of the fungus body, (b) is the time change of the angle θ and the distance d from the free surface, and (c) is the angle φ and the distance d from the free surface. The time change is shown. Although the size of Vibrio is as small as several micrometers, it can be seen from FIGS. 4 and 5 that information on the three-dimensional position and rotation can be obtained.
本実施例においては、大きさ数マイクロメートルの対象物を観察するために拡大倍率が50倍程度の対物レンズを用いたが、数十マイクロメートル立方の領域の観察を、0.5マイクロメートルの空間解像度をもって行うことが可能である。 In the present embodiment, an objective lens having an enlargement magnification of about 50 times is used to observe an object having a size of several micrometers, but observation of an area of several tens of micrometers cubic is 0.5 micrometers. It can be done with spatial resolution.
本発明の応用例の一つに、自由空間内における精子の運動観察が挙げられる。精子の遊泳軌跡はらせん形であり、その特徴を捉えるには三次元観察が不可欠である。また、精子の大きさは10マイクロメートル程度、遊泳軌跡のらせん半径は数十マイクロメートルであることから、本発明が有用であることが期待される。また、ブラウン運動が境界から受ける影響や、発酵などにともなう溶液中の微小気泡の観察などにも応用できる。 One application example of the present invention is observation of sperm movement in free space. The sperm swimming trajectory is helical, and three-dimensional observation is indispensable to capture its features. Further, since the size of the sperm is about 10 micrometers and the spiral radius of the swimming locus is several tens of micrometers, the present invention is expected to be useful. It can also be applied to the effects of Brownian motion from the boundary and observation of microbubbles in the solution accompanying fermentation.
1 透過光源
2 位相差コンデンサ
3 対物レンズ
4 対物レンズ位置調節ハンドル
5 CCDカメラ
6 透過光源
7 位相差コンデンサ
8 対物レンズ
9 xyz調節台
10 CCDカメラ
11 サンプルホルダ
12 xy調節台
13 光路折り曲げミラー
14 ビデオマイクロスコープ
15 サンプルホルダ
16 ガラス
17 ガラス
18 対物レンズ
19 対物レンズ
20 試料溶液
DESCRIPTION OF SYMBOLS 1 Transmission light source 2 Phase difference condenser 3 Objective lens 4 Objective lens position adjustment handle 5 CCD camera 6 Transmission light source 7 Phase difference condenser 8 Objective lens 9 xyz adjustment stand 10 CCD camera 11 Sample holder 12 xy adjustment stand 13 Optical path bending mirror 14 Video micro Scope 15 Sample holder 16 Glass 17 Glass 18 Objective lens 19 Objective lens 20 Sample solution
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