JP2005345561A - Scanning type laser microscope device - Google Patents

Scanning type laser microscope device Download PDF

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JP2005345561A
JP2005345561A JP2004162288A JP2004162288A JP2005345561A JP 2005345561 A JP2005345561 A JP 2005345561A JP 2004162288 A JP2004162288 A JP 2004162288A JP 2004162288 A JP2004162288 A JP 2004162288A JP 2005345561 A JP2005345561 A JP 2005345561A
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Nobuhiro Kita
信浩 北
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Olympus Corp
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<P>PROBLEM TO BE SOLVED: To provide a scanning type laser microscope device with which a fluorescence image having a good contrast can be obtained with simple constitution. <P>SOLUTION: A scanning type laser microscope device 100 which emits a laser beam from a laser beam source 10 and scans a sample 63 by irradiating the sample 63 with the laser beam and obtains a fluorescence image by detecting fluorescence emitted from the sample 63 is provided with a polarizing beams splitter 4 and a quarter-wave plate 61 which are arranged on the optical path between the laser beam source 10 and the sample 63, and which separate the laser beam reflected from the sample 63 and fluorescence emitted from the sample 63 by difference in polarized states. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は、試料にレーザ光を照射しつつ走査し、試料から発する蛍光を検出して蛍光画像を取得する走査型レーザ顕微鏡装置に関するものである。   The present invention relates to a scanning laser microscope apparatus that scans a sample while irradiating the sample with laser light, detects fluorescence emitted from the sample, and acquires a fluorescence image.

従来、励起光であるレーザ光を試料に照射しつつ走査し、試料から発する蛍光画像を取得する走査型レーザ顕微鏡が多用されている。ところで、近年、蛍光試薬の種類の増加とともに様々な検出ファクターの測定が望まれている。この検出ファクターとして、蛍光色素毎の蛍光画像の取得、蛍光スペクトルの定量化等がある。このような種々の検出ファクターを測定するには、試料から発する蛍光のみを抽出し、試料で反射される励起光を排除する必要がある。そのため、光の回折効果を利用して励起光と蛍光とを分離する音響光学素子（ＡＯＴＦ：Acousto-Optic Tunable Filter）を用いる方法が提示されている（特許文献１参照）。   Conventionally, a scanning laser microscope that scans while irradiating a laser beam, which is excitation light, and acquires a fluorescent image emitted from the sample is widely used. By the way, in recent years, measurement of various detection factors is desired along with an increase in types of fluorescent reagents. This detection factor includes acquisition of a fluorescence image for each fluorescent dye, quantification of a fluorescence spectrum, and the like. In order to measure such various detection factors, it is necessary to extract only the fluorescence emitted from the sample and exclude the excitation light reflected by the sample. Therefore, a method using an acousto-optic tunable filter (AOTF: Acousto-Optic Tunable Filter) that separates excitation light and fluorescence using a light diffraction effect has been proposed (see Patent Document 1).

特開２００３−１７７３２号公報JP 2003-17732 A

しかしながら、ＡＯＴＦは、波長の差によって回折分光し、励起光と蛍光とを分離するが、励起光の光強度が蛍光の光強度に比して非常に強いため、回折分光した蛍光成分に励起光成分が漏れ入る。蛍光の光強度は、励起光の光強度に比して微弱であるため、蛍光成分に励起光成分が漏れ入ると、コントラストが悪い蛍光画像しか取得できないという問題点があった。   However, AOTF performs diffraction spectroscopy by the difference in wavelength and separates excitation light and fluorescence. However, since the light intensity of excitation light is much stronger than the light intensity of fluorescence, excitation light is added to the fluorescence component subjected to diffraction spectroscopy. Ingredients leak. Since the fluorescence light intensity is weaker than the excitation light intensity, if the excitation light component leaks into the fluorescence component, there is a problem that only a fluorescence image with poor contrast can be acquired.

本発明は、上記に鑑みてなされたものであって、簡易な構成によって試料で反射される励起光を排除し、コントラストの良い蛍光画像が取得できる走査型レーザ顕微鏡装置を提供することを目的とする。   The present invention has been made in view of the above, and an object of the present invention is to provide a scanning laser microscope apparatus capable of eliminating excitation light reflected by a sample with a simple configuration and acquiring a fluorescent image with good contrast. To do.

上記目的を達成するために、請求項１にかかる走査型レーザ顕微鏡装置は、レーザ光源からレーザ光を出射し、前記レーザ光を試料に照射しつつ走査し、前記試料から発する蛍光を検出して蛍光画像を取得する走査型レーザ顕微鏡装置において、前記レーザ光源と前記試料との間の光路上に配置され、前記試料から反射された前記レーザ光と前記試料から発した蛍光とを偏光状態の違いによって分離する分離手段を備えたことを特徴とする。   In order to achieve the above object, a scanning laser microscope apparatus according to claim 1 emits a laser beam from a laser light source, scans the sample while irradiating the sample with the laser beam, and detects fluorescence emitted from the sample. In a scanning laser microscope apparatus for acquiring a fluorescence image, a difference in polarization state between the laser light reflected from the sample and the fluorescence emitted from the sample is disposed on the optical path between the laser light source and the sample. Separating means for separating by the above is provided.

また、請求項２にかかる走査型レーザ顕微鏡装置は、上記の発明において、前記分離手段は、偏光ビームスプリッタと１／４波長板との組合わせであることを特徴とする。   The scanning laser microscope apparatus according to claim 2 is characterized in that, in the above invention, the separating means is a combination of a polarizing beam splitter and a quarter wavelength plate.

また、請求項３にかかる走査型レーザ顕微鏡装置は、上記の発明において、前記光路上に配置され、光の波長によって回折分光を行う回折手段をさらに備えたことを特徴とする。   According to a third aspect of the present invention, there is provided the scanning laser microscope apparatus according to the above invention, further comprising a diffractive means disposed on the optical path and performing diffraction spectroscopy according to the wavelength of light.

また、請求項４にかかる走査型レーザ顕微鏡装置は、上記の発明において、前記回折手段は、音響光学素子であることを特徴とする。   The scanning laser microscope apparatus according to a fourth aspect of the present invention is characterized in that, in the above invention, the diffractive means is an acousto-optic element.

本発明にかかる走査型レーザ顕微鏡装置は、偏光ビームスプリッタと１／４波長板とを組合わせて光路上に配置することによって、簡易な構成でコントラストの良い蛍光画像が取得できるという効果を奏する。   The scanning laser microscope apparatus according to the present invention has an effect that a fluorescent image having a good contrast can be obtained with a simple configuration by combining a polarizing beam splitter and a quarter-wave plate on the optical path.

以下に添付図面を参照して、この発明にかかる走査型レーザ顕微鏡装置の好適な実施の形態を詳細に説明する。   Exemplary embodiments of a scanning laser microscope apparatus according to the present invention will be explained below in detail with reference to the accompanying drawings.

（実施の形態）
図１は、この発明の実施の形態である走査型レーザ顕微鏡装置１００の概要構成を示すブロック図である。図１において、この走査型レーザ顕微鏡装置１００は、光源部１と、音響光学素子（ＡＯＴＦ）２と、共焦点光学部３と、偏光ビームスプリッタ４と、走査光学部５と、顕微鏡部６と、検出器７Ａ，７Ｂと、信号処理部８と、制御部９とを有している。さらに、光源部１は、レーザ光源１０と、コリメータレンズ１１とを有し、共焦点光学部３は、共焦点レンズ３０と、共焦点ピンホール３１と、共焦点レンズ３２とを有し、走査光学部５は、ガルバノミラー５０を有し、顕微鏡部６は、結像レンズ６０と、１／４波長板６１と、対物レンズ６２とを有している。 (Embodiment)
FIG. 1 is a block diagram showing a schematic configuration of a scanning laser microscope apparatus 100 according to an embodiment of the present invention. In FIG. 1, the scanning laser microscope apparatus 100 includes a light source unit 1, an acoustooptic device (AOTF) 2, a confocal optical unit 3, a polarization beam splitter 4, a scanning optical unit 5, and a microscope unit 6. , Detectors 7A and 7B, a signal processing unit 8, and a control unit 9. Further, the light source unit 1 includes a laser light source 10 and a collimator lens 11, and the confocal optical unit 3 includes a confocal lens 30, a confocal pinhole 31, and a confocal lens 32, and scanning. The optical unit 5 includes a galvanometer mirror 50, and the microscope unit 6 includes an imaging lens 60, a quarter wavelength plate 61, and an objective lens 62.

まず、レーザ光源１０から励起光である波長５８０ｎｍのレーザ光が出射され、出射されたレーザ光は、ＡＯＴＦ２、共焦点光学部３、偏光ビームスプリッタ４、走査光学部５を順次介して顕微鏡部６に入射する。顕微鏡部６に入射したレーザ光は、結像レンズ６０、１／４波長板６１、対物レンズ６２を順次介して試料６３に入射する。試料６３に入射したレーザ光は、試料６２を励起し、試料６２から波長６００ｎｍの蛍光が発光する。蛍光は、対物レンズ６２、１／４波長板６１、結像レンズ６０を順次介して顕微鏡部６から出射し、走査光学部５、偏光ビームスプリッタ４、共焦点光学部３を順次介してＡＯＴＦ２に入射する。ＡＯＴＦ２に入射した蛍光は、±１次に回折分光され、＋１次に回折分光された蛍光は、検出器７Ａに入射し、−１次に回折分光された蛍光は、検出器７Ｂに入射する。検出器７Ａ，７Ｂは、入射した蛍光を電気信号に変換して信号処理部８に出力する。信号処理部８は、入力した電気信号を加算し、加算した加算信号を制御部９に出力する。制御部９は、入力した加算信号を画像信号に変換し、表示部１２に出力し、表示部１２は、入力した画像信号を蛍光画像として出力表示する。   First, laser light having a wavelength of 580 nm, which is excitation light, is emitted from the laser light source 10, and the emitted laser light sequentially passes through the AOTF 2, the confocal optical unit 3, the polarization beam splitter 4, and the scanning optical unit 5. Is incident on. The laser light incident on the microscope unit 6 is incident on the sample 63 through the imaging lens 60, the quarter wavelength plate 61, and the objective lens 62 in this order. The laser light incident on the sample 63 excites the sample 62, and fluorescence having a wavelength of 600 nm is emitted from the sample 62. Fluorescence is emitted from the microscope unit 6 through the objective lens 62, the quarter-wave plate 61, and the imaging lens 60 in order, and is sequentially transmitted to the AOTF 2 through the scanning optical unit 5, the polarizing beam splitter 4, and the confocal optical unit 3. Incident. The fluorescence that has entered the AOTF 2 is diffracted and spectrumd in the ± 1st order, the fluorescence that has been diffracted and spectrumd in the + 1st order is incident on the detector 7A, and the fluorescence that has been diffracted and spectrumd in the −1st order is incident on the detector 7B. The detectors 7A and 7B convert the incident fluorescence into an electrical signal and output it to the signal processing unit 8. The signal processing unit 8 adds the input electrical signals and outputs the added addition signal to the control unit 9. The control unit 9 converts the input addition signal into an image signal and outputs it to the display unit 12, and the display unit 12 outputs and displays the input image signal as a fluorescent image.

ところで、試料６３を照射したレーザ光の大部分は、試料６３を透過するが、一部は、試料６３で反射され、試料６３から発した蛍光とともにＡＯＴＦ２まで逆行する。ＡＯＴＦ２は、入射した光を回折現象によって回折分光し、回折分光する光の波長を可変できる機能を有している。したがって、複数の蛍光色素で染色した試料６３から複数の異なる波長の蛍光を同時に選別し、蛍光色素毎に蛍光画像を取得する場合に多用される。図２は、ＡＯＴＦ２の回折効率ηを示す特性図である。図２に示すように、例えば蛍光波長λ０が６００ｎｍであり、蛍光波長λ０の回折効率ηEMを１とした場合、励起光波長５８０ｎｍの回折効率ηEXは、１／１００となる。したがって、ＡＯＴＦ２は、蛍光を抽出するものの、蛍光に対して１／１００の比で励起光も抽出することになる。励起光の光強度は、蛍光の光強度に対して非常に強いため、励起光の回折効率ηEXが蛍光の回折効率ηEMの１／１００になったとしても、蛍光画像のコントラストを悪化させる要因になる。 By the way, most of the laser light irradiated to the sample 63 is transmitted through the sample 63, but a part of the laser light is reflected by the sample 63 and goes back to AOTF2 together with the fluorescence emitted from the sample 63. The AOTF 2 has a function of diffracting the incident light by a diffraction phenomenon and changing the wavelength of the light to be diffracted. Therefore, it is frequently used when a plurality of fluorescent lights having different wavelengths are simultaneously selected from the sample 63 stained with a plurality of fluorescent dyes and a fluorescent image is acquired for each fluorescent dye. FIG. 2 is a characteristic diagram showing the diffraction efficiency η of AOTF2. As shown in FIG. 2, for example, a fluorescence wavelength .lambda.0 is 600 nm, when the 1 diffraction efficiency eta EM fluorescence wavelength .lambda.0, the diffraction efficiency eta EX of the excitation light wavelength 580nm is 1/100. Therefore, although AOTF2 extracts fluorescence, excitation light is also extracted at a ratio of 1/100 with respect to fluorescence. Since the light intensity of the excitation light is very high with respect to the light intensity of the fluorescence, even if the diffraction efficiency η EX of the excitation light becomes 1/100 of the diffraction efficiency η EM of the fluorescence, the contrast of the fluorescence image is deteriorated. It becomes a factor.

図３は、偏光ビームスプリッタ４と１／４波長板６１とを組合わせて配置することによって、ＡＯＴＦ２に入射する前の段階で励起光と蛍光とを分離する過程を示す模式図である。図３において、試料６３に入射する励起光を入射励起光Ａ１とし、試料６３で反射された励起光を反射励起光Ａ２とし、試料６３から発した蛍光を蛍光Ｂとした場合、まず、レーザ光源１０から出射されたレーザ光は、入射励起光Ａ１として偏光ビームスプリッタ４に入射する。入射励起光Ａ１は、直線偏光であり、入射励起光Ａ１の偏光方向は、偏光ビームスプリッタ４を透過するように整合されている。このため、入射励起光Ａ１は、偏光ビームスプリッタ４を透過し、１／４波長板６１に入射する。また、１／４波長板６１の向きは、入射励起光Ａ１を右回転の円偏光に変換するように整合されている。このため、入射励起光Ａ１は、１／４波長板６１によって、右回転の円偏光に変換され、試料６３に入射する。試料６３に入射した入射励起光Ａ１の大部分は、試料６３に吸収されるが、一部は、試料６３で反射され、反射励起光Ａ２となる。   FIG. 3 is a schematic diagram showing a process of separating excitation light and fluorescence before entering the AOTF 2 by arranging the polarization beam splitter 4 and the quarter-wave plate 61 in combination. In FIG. 3, when the excitation light incident on the sample 63 is incident excitation light A1, the excitation light reflected by the sample 63 is reflected excitation light A2, and the fluorescence emitted from the sample 63 is fluorescence B, first, a laser light source The laser light emitted from 10 enters the polarization beam splitter 4 as incident excitation light A1. The incident excitation light A1 is linearly polarized light, and the polarization direction of the incident excitation light A1 is aligned so as to pass through the polarization beam splitter 4. For this reason, the incident excitation light A <b> 1 passes through the polarization beam splitter 4 and enters the quarter wavelength plate 61. The direction of the quarter-wave plate 61 is matched so as to convert the incident excitation light A1 into right-handed circularly polarized light. For this reason, the incident excitation light A 1 is converted into right-handed circularly polarized light by the quarter-wave plate 61 and enters the sample 63. Most of the incident excitation light A1 incident on the sample 63 is absorbed by the sample 63, but a part is reflected by the sample 63 and becomes reflected excitation light A2.

反射励起光Ａ２は、試料６３で反射されると、円偏光の回転方向が反転し、左回転の円偏光となる。左回転の円偏光となった反射励起光Ａ２は、１／４波長板６１に入射する。１／４波長板６１は、入射した反射励起光Ａ２を直線偏光に変換する。したがって、１／４波長板６１に入射した入射励起光Ａ１と１／４波長板６１から出射する反射励起光Ａ２との位相差は、π／２になり、入射励起光Ａ１と反射励起光Ａ２との偏光角は、９０°異なる。したがって、反射励起光Ａ２は、入射励起光Ａ１と９０°異なった直線偏光光となって偏光ビームスプリッタ４に入射する。偏光ビームスプリッタ４は、入射励起光Ａ１が透過するように整合されているため、偏光角が入射励起光Ａ１と９０°異なる反射励起光Ａ２を反射する。この結果、偏光ビームスプリッタ４まで入射励起光Ａ１と同じ光路を逆行した反射励起光Ａ２は、偏光ビームスプリッタ４によって分離される。しかし、上述の原理にも係わらず、偏光ビームスプリッタ４を透過する反射励起光Ａ２が存在し、反射励起光Ａ２の反射と透過との比率は、一般的に１：１／１００〜１／５００程度である。   When the reflected excitation light A2 is reflected by the sample 63, the rotation direction of the circularly polarized light is reversed to become counterclockwise circularly polarized light. The reflected excitation light A <b> 2 that has become counterclockwise circularly polarized light enters the quarter-wave plate 61. The quarter wave plate 61 converts the incident reflected excitation light A2 into linearly polarized light. Therefore, the phase difference between the incident excitation light A1 incident on the quarter-wave plate 61 and the reflected excitation light A2 emitted from the quarter-wave plate 61 is π / 2, and the incident excitation light A1 and the reflected excitation light A2 And the polarization angle differ by 90 °. Therefore, the reflected excitation light A2 enters the polarization beam splitter 4 as linearly polarized light that is 90 ° different from the incident excitation light A1. Since the polarization beam splitter 4 is aligned so as to transmit the incident excitation light A1, it reflects the reflected excitation light A2 whose polarization angle is 90 ° different from that of the incident excitation light A1. As a result, the reflected excitation light A2 that travels backward along the same optical path as the incident excitation light A1 up to the polarization beam splitter 4 is separated by the polarization beam splitter 4. However, in spite of the above-described principle, there is reflected excitation light A2 that passes through the polarizing beam splitter 4, and the ratio between reflection and transmission of the reflected excitation light A2 is generally 1: 1/100 to 1/500. Degree.

また、図３において、入射励起光Ａ１の照射によって試料６３は励起され、試料６３から蛍光が発せられる。試料６３から発した蛍光を蛍光Ｂとした場合、蛍光Ｂは、ランダム偏光であり、１／４波長板６１を通過しても、ランダム偏光に変化なく、偏光ビームスプリッタ４に入射する。偏光ビームスプリッタ４は、入射した蛍光Ｂを直交するＰ偏光成分の光とＳ偏光成分の光とに分離し、Ｓ偏光成分の光を反射し、Ｐ偏光成分の光を透過する。蛍光Ｂは、ランダム偏光であるため、偏光ビームスプリッタ４によって、蛍光Ｂの１／２は、透過蛍光Ｂ１となり、蛍光Ｂの１／２は、反射蛍光Ｂ２となる。したがって、偏光ビームスプリッタ４を透過し、ＡＯＴＦ２に入射する蛍光Ｂの入射比率は、１／２となる。   In FIG. 3, the sample 63 is excited by irradiation with the incident excitation light A <b> 1, and fluorescence is emitted from the sample 63. When the fluorescence emitted from the sample 63 is fluorescence B, the fluorescence B is randomly polarized light, and is incident on the polarization beam splitter 4 without changing to random polarized light even when passing through the quarter-wave plate 61. The polarization beam splitter 4 separates the incident fluorescence B into orthogonal P-polarized component light and S-polarized component light, reflects S-polarized component light, and transmits P-polarized component light. Since the fluorescence B is random polarization, ½ of the fluorescence B becomes the transmitted fluorescence B1 and ½ of the fluorescence B becomes the reflected fluorescence B2 by the polarization beam splitter 4. Therefore, the incidence ratio of the fluorescence B that passes through the polarizing beam splitter 4 and enters the AOTF 2 is ½.

つまり、試料６３で反射された励起光は、偏光ビームスプリッタ４によって、１／１００〜１／５００に減衰され、ＡＯＴＦ２によって、さらに１／１００に減衰される。したがって、試料６３で反射された励起光は、１／１０，０００〜１／５０，０００に減衰されて検出器７Ａ，７Ｂに入射し、蛍光は、偏光ビームスプリッタ４によって１／２に減衰されて検出器７Ａ，７Ｂに入射する。   That is, the excitation light reflected by the sample 63 is attenuated to 1/100 to 1/500 by the polarization beam splitter 4, and further attenuated to 1/100 by the AOTF2. Therefore, the excitation light reflected by the sample 63 is attenuated by 1 / 10,000 to 1 / 50,000 and enters the detectors 7A and 7B, and the fluorescence is attenuated by 1/2 by the polarization beam splitter 4. Incident on the detectors 7A and 7B.

この結果、検出器７Ａ，７Ｂに入射する蛍光と励起光との入射比率は、１／２：１／１０，０００〜１／２：５０，０００＝１：１／５，０００〜１：１／２５，０００となる。従来、検出器７Ａ，７Ｂに入射する蛍光と励起光との入射比率は、１：１／１００であることから、この発明によれば、蛍光が相対的に５０〜２５０倍明るくなり、コントラストの良い蛍光画像が取得できる。   As a result, the incident ratio of fluorescence and excitation light incident on the detectors 7A and 7B is 1/2: 1 / 10,000 to 1/2: 50,000 = 1: 1 / 5,000 to 1: 1. / 25,000. Conventionally, since the incident ratio of fluorescence and excitation light incident on the detectors 7A and 7B is 1: 1/100, according to the present invention, the fluorescence becomes relatively 50 to 250 times brighter and the contrast is increased. A good fluorescence image can be acquired.

この実施の形態では、偏光ビームスプリッタ４と１／４波長板６１とを組み合わせてレーザ光上に配置することによって、検出器７Ａ，７Ｂに入射する励起光を減衰させ、コントラストのよい蛍光画像が取得できるようにしている。   In this embodiment, the polarization beam splitter 4 and the quarter-wave plate 61 are combined and disposed on the laser light, thereby attenuating the excitation light incident on the detectors 7A and 7B, thereby producing a fluorescent image with good contrast. You can get it.

なお、この実施の形態では、偏光ビームスプリッタ４を透過した蛍光を検出器７Ａ，７Ｂに入射するようにして、偏光ビームスプリッタ４を透過する反射励起光Ａ２を減衰するようにしていたが、偏光ビームスプリッタ４で反射された蛍光を検出器７Ａ，７Ｂに入射し、偏光ビームスプリッタ４で反射される反射励起光Ａ２を減衰するようにしてもよい。また、１／４波長板６１は、入射した入射励起光Ａ１を右回転の円偏光に変換していたが、左回転の円偏光に変換するようにしてもよい。   In this embodiment, the fluorescence transmitted through the polarization beam splitter 4 is incident on the detectors 7A and 7B, and the reflected excitation light A2 transmitted through the polarization beam splitter 4 is attenuated. The fluorescence reflected by the beam splitter 4 may enter the detectors 7A and 7B, and the reflected excitation light A2 reflected by the polarization beam splitter 4 may be attenuated. Further, although the ¼ wavelength plate 61 converts the incident incident excitation light A1 into right-handed circularly polarized light, it may be converted into left-handed circularly polarized light.

この発明の実施の形態にかかる走査型レーザ顕微鏡装置を示す機能ブロック図である。1 is a functional block diagram showing a scanning laser microscope apparatus according to an embodiment of the present invention. この発明の実施の形態にかかるＡＯＴＦの特性を示すグラフである。It is a graph which shows the characteristic of AOTF concerning an embodiment of this invention. この発明の実施の形態にかかる偏光ビームスプリッタと１／４波長板との働きを示す模式図である。It is a schematic diagram which shows the function of the polarizing beam splitter and quarter wave plate concerning embodiment of this invention.

符号の説明Explanation of symbols

１ 光源部
２ ＡＯＴＦ
３ 共焦点光学部
４ 偏光ビームスプリッタ
５ 走査光学部
６ 顕微鏡部
７Ａ，７Ｂ 検出器
８ 信号処理部
９ 制御部
１０ レーザ光源
１１ コリメータレンズ
１２ 表示部
３０ レンズ
３１ 共焦点ピンホール
３２ 共焦点レンズ
５１ ガルバノミラー
６０ 結像レンズ
６１ １／４波長板
６２ 対物レンズ
６３ 試料
１００ 走査型レーザ顕微鏡装置 1 Light source 2 AOTF
DESCRIPTION OF SYMBOLS 3 Confocal optical part 4 Polarization beam splitter 5 Scanning optical part 6 Microscope part 7A, 7B Detector 8 Signal processing part 9 Control part 10 Laser light source 11 Collimator lens 12 Display part 30 Lens 31 Confocal pinhole 32 Confocal lens 51 Galvano Mirror 60 Imaging lens 61 1/4 wavelength plate 62 Objective lens 63 Sample 100 Scanning laser microscope apparatus

Claims (4)

レーザ光源からレーザ光を出射し、前記レーザ光を試料に照射しつつ走査し、前記試料から発する蛍光を検出して蛍光画像を取得する走査型レーザ顕微鏡装置において、
前記レーザ光源と前記試料との間の光路上に配置され、前記試料から反射された前記レーザ光と前記試料から発した蛍光とを偏光状態の違いによって分離する分離手段を備えたことを特徴とする走査型レーザ顕微鏡装置。 In a scanning laser microscope apparatus that emits laser light from a laser light source, scans while irradiating the sample with the laser light, detects fluorescence emitted from the sample, and acquires a fluorescence image.
A separation unit disposed on an optical path between the laser light source and the sample, and separating the laser light reflected from the sample and fluorescence emitted from the sample according to a difference in polarization state; Scanning laser microscope apparatus. 前記分離手段は、偏光ビームスプリッタと１／４波長板との組合わせであることを特徴とする請求項１に記載の走査型レーザ顕微鏡装置。   2. The scanning laser microscope apparatus according to claim 1, wherein the separating means is a combination of a polarizing beam splitter and a quarter wave plate. 前記光路上に配置され、光の波長によって回折分光を行う回折手段をさらに備えたことを特徴とする請求項１または２に記載の走査型レーザ顕微鏡装置。   The scanning laser microscope apparatus according to claim 1, further comprising a diffracting unit disposed on the optical path and performing diffraction spectroscopy according to a wavelength of light. 前記回折手段は、音響光学素子であることを特徴とする請求項３に記載の走査型レーザ顕微鏡装置。   The scanning laser microscope apparatus according to claim 3, wherein the diffracting means is an acousto-optic element.
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