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- 238000001514 detection method Methods 0.000 claims description 34
- 239000000126 substance Substances 0.000 claims description 22
- 238000005286 illumination Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 11
- 230000001678 irradiating Effects 0.000 claims description 9
- 230000000051 modifying Effects 0.000 claims description 8
- 210000001747 Pupil Anatomy 0.000 claims description 7
- 230000003287 optical Effects 0.000 claims 1
- 230000000875 corresponding Effects 0.000 description 1
Description
本発明の第１の態様においては、顕微鏡が提供される。顕微鏡は、第１対物レンズを含み、試料に含まれる蛍光物質を活性化させる第１光と、第１光と異なり活性化された蛍光物質を励起する第２光とを、試料に対して第１方向から照射する照明系を備える。顕微鏡は、第２対物レンズを含み、第１光と第２光とが照射された試料から放射される蛍光を、第１方向とは異なる第２方向から検出する検出系を備える。顕微鏡は、第１対物レンズの瞳面における第１光の強度分布と第２光の強度分布とが互いに異なるように、第１光及び第２光の少なくとも一方を成形するビーム成形部を備える。 Oite to a first aspect of the present invention, the microscope is provided. Microscope, it includes a first objective lens, and the first light Ru activate the fluorescent substance contained in the sample, and a second light you excite the fluorescent substance activated which Unlike the first light, the sample On the other hand, it is provided with an illumination system that illuminates from the first direction . Microscope includes a second objective lens, a fluorescence the first light and second light is emitted from the sample irradiated, Ru comprising a detection system for detecting the second direction different from the first direction. The microscope includes a beam forming portion that forms at least one of the first light and the second light so that the intensity distribution of the first light and the intensity distribution of the second light on the pupil surface of the first objective lens are different from each other.
本発明の第２の態様においては、顕微鏡が提供される。顕微鏡は、試料に含まれる蛍光物質を励起する第１光と、第１光と異なり、励起された蛍光物質に誘導放出を生じさせる第２光とを、試料に対して第１方向から照射する照明系を備える。顕微鏡は、試料から放射される蛍光を、第１方向とは異なる第２方向から検出する検出系を備える。顕微鏡は、制御部を備える。制御部は、照明系及び検出系を制御して、第１光のみを試料に照射し、試料から放射される第１の蛍光を検出する第１検出処理と、第１光及び第２光を試料に照射し、試料から照射される第２の蛍光を検出する第２検出処理とを順次実施する。本発明の第３の態様においては、顕微鏡が提供される。顕微鏡は、試料に含まれる蛍光物質を励起する第１光と、第１光と異なり、励起された蛍光物質に誘導放出を生じさせる第２光とを、試料に対して第１方向から照射する照明系を備える。顕微鏡は、試料から放射される蛍光を、第１方向とは異なる第２方向から検出する検出系を備える。顕微鏡は、第１光の強度と第２光の強度とを互いに異なる周波数で変調する変調部を備える。検出系は、蛍光を検出するロックイン検出部を有する。 Oite to a second aspect of the present invention, the microscope is provided. Microscope, a first light you excite the fluorescent substance contained in the sample, unlike the first light and a second light that generates stimulated emission in the excited fluorescent substance, a first direction relative to the sample It is equipped with an illumination system that illuminates from . Microscope, the fluorescence emitted from the specimen, Ru comprising a detection system for detecting the second direction different from the first direction. The microscope includes a control unit. The control unit controls the illumination system and the detection system to irradiate the sample with only the first light, and performs the first detection process of detecting the first fluorescence emitted from the sample, and the first light and the second light. The second detection process of irradiating the sample and detecting the second fluorescence emitted from the sample is sequentially performed. In a third aspect of the invention, a microscope is provided. The microscope irradiates the sample with the first light that excites the fluorescent substance contained in the sample and the second light that causes stimulated emission of the excited fluorescent substance unlike the first light from the first direction. Equipped with a lighting system. The microscope includes a detection system that detects the fluorescence emitted from the sample from a second direction different from the first direction. The microscope includes a modulator that modulates the intensity of the first light and the intensity of the second light at different frequencies. The detection system has a lock-in detection unit that detects fluorescence.
本発明の第４の態様においては、観察方法が提供される。観察方法は、第１対物レンズを含む照明系が、試料に含まれる蛍光物質を活性化させる第１光と、第１光と異なり活性化された蛍光物質を励起する第２光とを、試料に対して第１方向から照射することを含む。観察方法は、第２対物レンズを含む検出系が、第１光と第２光とが照射された試料から放射される蛍光を、第１方向とは異なる第２方向から検出することを含む。観察方法は、ビーム成形部が、第１対物レンズの瞳面における第１光の強度分布と第２光の強度分布とが互いに異なるように、第１光及び第２光の少なくとも一方を成形することを含む。 Oite to a fourth aspect of the present invention, the observation method is provided. Observation method, the illumination system including the first objective lens, the first light Ru activate a fluorescent material contained in the sample, and the second light you excite the fluorescent substance activated which Unlike the first light Includes irradiating the sample from the first direction . Observation method, the detection system comprising a second objective lens, a fluorescence emitted from a sample and the first light and second light is irradiated, including that you detect the second direction different from the first direction Mmm. In the observation method, the beam forming portion molds at least one of the first light and the second light so that the intensity distribution of the first light and the intensity distribution of the second light on the pupil surface of the first objective lens are different from each other. Including that.
本発明の第５の態様においては、観察方法が提供される。観察方法は、照明系が、試料に含まれる蛍光物質を励起する第１光と、第１光と異なり、励起された蛍光物質に誘導放出を生じさせる第２光とを、試料に対して第１方向から照射することを含む。観察方法は、検出系が、試料から放射される蛍光を、第１方向とは異なる第２方向から検出することを含む。観察方法は、制御部が、照明系及び検出系を制御して、第１光のみを試料に照射し、試料から放射される第１の蛍光を検出する第１検出処理と、第１光及び第２光を試料に照射し、試料から照射される第２の蛍光を検出する第２検出処理とを順次実施することを含む。本発明の第６の態様においては、観察方法が提供される。観察方法は、照明系が、試料に含まれる蛍光物質を励起する第１光と、第１光と異なり、励起された蛍光物質に誘導放出を生じさせる第２光とを、試料に対して第１方向から照射することを含む。観察方法は、蛍光を検出するロックイン検出部を有する検出系が、試料から放射される蛍光を、第１方向とは異なる第２方向から検出することを含む。観察方法は、変調部が、第１光の強度と第２光の強度とを互いに異なる周波数で変調することを含む。 Oite to a fifth aspect of the present invention, the observation method is provided. Observation method, the illumination system, the first light you excite the fluorescent substance contained in the sample, unlike the first light and a second light that generates stimulated emission in the excited fluorescent material, a sample On the other hand, it includes irradiating from the first direction . Observation method, the detection system, the fluorescence emitted from the sample, including that you detect the second direction different from the first direction. The observation method includes a first detection process in which the control unit controls the illumination system and the detection system, irradiates the sample with only the first light, and detects the first fluorescence emitted from the sample, and the first light and the first light. This includes sequentially performing a second detection process of irradiating the sample with the second light and detecting the second fluorescence emitted from the sample. In the sixth aspect of the present invention, an observation method is provided. In the observation method, the illumination system emits the first light that excites the fluorescent substance contained in the sample and the second light that causes stimulated emission of the excited fluorescent substance unlike the first light. It includes irradiating from one direction. The observation method includes a detection system having a lock-in detection unit for detecting fluorescence, which detects the fluorescence emitted from the sample from a second direction different from the first direction. The observation method includes that the modulation unit modulates the intensity of the first light and the intensity of the second light at different frequencies from each other.
ステップＳ３５において、顕微鏡１は、ステップＳ３２の検出結果とステップＳ３４の検出結果とに基づいて、画像を生成する。例えば、顕微鏡１（例、画像処理部）は、ステップＳ３２の第１検出処理で得られる第１画像と、ステップＳ３４の第２検出処理で得られる第２画像との比較によって第３画像（結果画像）を生成する。例えば、顕微鏡１は、第３画像として、第１画像と第２画像との差分に相当する画像を生成する。 In step S35, the microscope 1, based on the detection results of the steps S 34 in step S32, to generate an image. For example, the microscope 1 (eg, the image processing unit) compares the first image obtained in the first detection process in step S32 with the second image obtained in the second detection process in step S34 to obtain a third image (result). Image) is generated. For example, the microscope 1 generates an image corresponding to the difference between the first image and the second image as the third image.
Claims (15)
第２対物レンズを含み、前記第１光と前記第２光とが照射された前記試料から放射される蛍光を、前記第１方向とは異なる第２方向から検出する検出系と、
前記第１対物レンズの瞳面における前記第１光の強度分布と前記第２光の強度分布とが互いに異なるように、前記第１光及び前記第２光の少なくとも一方を成形するビーム成形部と、を備える顕微鏡。 It includes a first objective lens, and the first light Ru activate the fluorescent substance contained in the sample, and a second light you exciting pre Symbol the fluorescent substance Ri activated different from the first light, the sample And the illumination system that illuminates from the first direction
A detection system including a second objective lens that detects fluorescence emitted from the sample irradiated with the first light and the second light from a second direction different from the first direction.
A beam forming portion that forms at least one of the first light and the second light so that the intensity distribution of the first light and the intensity distribution of the second light on the pupil surface of the first objective lens are different from each other. A microscope equipped with.
前記試料から放射される蛍光を、前記第１方向とは異なる第２方向から検出する検出系と、
制御部と、を備え、
前記制御部は、前記照明系及び前記検出系を制御して、前記第１光のみを前記試料に照射し、前記試料から放射される第１の蛍光を検出する第１検出処理と、前記第１光及び前記第２光を前記試料に照射し、前記試料から照射される第２の蛍光を検出する第２検出処理とを順次実施する、顕微鏡。 A first light you excite the fluorescent substance contained in the sample, before Symbol Unlike the first light and a second light that generates stimulated emission in the excited the fluorescent substance, the relative to the sample 1 The lighting system that illuminates from the direction and
The fluorescence emitted from the front Symbol sample, a detection system for detecting the second direction different from the first direction,
And a control unit, a
The control unit controls the illumination system and the detection system, irradiates the sample with only the first light, and detects the first fluorescence emitted from the sample, and the first detection process. 1 light and irradiates the second light to the sample, it successively performed and a second detection process for detecting a second fluorescence emitted from the sample, the microscope.
前記試料から放射される蛍光を、前記第１方向とは異なる第２方向から検出する検出系と、
前記第１光の強度と前記第２光の強度とを互いに異なる周波数で変調する変調部と、を備え、
前記検出系は、前記蛍光を検出するロックイン検出部を有する、顕微鏡。 The sample is irradiated with the first light that excites the fluorescent substance contained in the sample and the second light that causes stimulated emission of the excited fluorescent substance unlike the first light from the first direction. Lighting system and
A detection system that detects the fluorescence emitted from the sample from a second direction different from the first direction,
A modulation unit that modulates the intensity of the first light and the intensity of the second light at different frequencies is provided.
The detection system, that have a lock-in detection unit for detecting a pre Kihotaru light microscope.
前記第１光及び前記第２光を、前記試料において前記第１方向及び前記第２方向のそれぞれと交差する方向に走査する走査部を備える、請求項１から請求項１０のいずれか一項に記載の顕微鏡。 The illumination system collects the first light and the second light on the sample.
Said first light and said second light, and a scanning unit for scanning in a direction intersecting with each of the first and second directions in said sample, any one of claims 1 0 to claim 1 The microscope described in.
第２対物レンズを含む検出系が、前記第１光と前記第２光とが照射された前記試料から放射される蛍光を、前記第１方向とは異なる第２方向から検出することと、
ビーム成形部が、前記第１対物レンズの瞳面における前記第１光の強度分布と前記第２光の強度分布とが互いに異なるように、前記第１光及び前記第２光の少なくとも一方を成形することと、を含む観察方法。 Illumination system comprising a first objective lens, the first light Ru activate the fluorescent substance contained in the sample, a pre-Symbol second light exciting the fluorescent substance Ri activated different from the first optical and irradiating the first direction with respect to the sample,
The detection system including the second objective lens detects the fluorescence emitted from the sample irradiated with the first light and the second light from a second direction different from the first direction.
The beam forming portion forms at least one of the first light and the second light so that the intensity distribution of the first light and the intensity distribution of the second light on the pupil surface of the first objective lens are different from each other. Observation methods including what to do .
検出系が、前記試料から放射される蛍光を、前記第１方向とは異なる第２方向から検出することと、
制御部が、前記照明系及び前記検出系を制御して、前記第１光のみを前記試料に照射し、前記試料から放射される第１の蛍光を検出する第１検出処理と、前記第１光及び前記第２光を前記試料に照射し、前記試料から照射される第２の蛍光を検出する第２検出処理とを順次実施することと、を含む観察方法。 Illumination system, a first light you excite the fluorescent substance contained in the sample, before Symbol Unlike the first light and a second light that generates stimulated emission in the excited the fluorescent substance, to the sample On the other hand, irradiating from the first direction and
The detection system detects the fluorescence emitted from the sample from a second direction different from the first direction.
The first detection process in which the control unit controls the illumination system and the detection system to irradiate the sample with only the first light and detect the first fluorescence emitted from the sample, and the first detection process. An observation method including sequentially performing a second detection process of irradiating the sample with light and the second light and detecting the second fluorescence emitted from the sample .
蛍光を検出するロックイン検出部を有する検出系が、前記試料から放射される前記蛍光を、前記第１方向とは異なる第２方向から検出することと、A detection system having a lock-in detection unit for detecting fluorescence detects the fluorescence emitted from the sample from a second direction different from the first direction.
変調部が、前記第１光の強度と前記第２光の強度とを互いに異なる周波数で変調することと、を含む観察方法。An observation method in which a modulation unit modulates the intensity of the first light and the intensity of the second light at frequencies different from each other.
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| US8570649B2 (en) * | 2009-10-29 | 2013-10-29 | California Institute Of Technology | Dual-mode raster point scanning/light sheet illumination microscope |
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