WO2018105026A1 - Laser scanning microscope - Google Patents

Laser scanning microscope Download PDF

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WO2018105026A1
WO2018105026A1 PCT/JP2016/086133 JP2016086133W WO2018105026A1 WO 2018105026 A1 WO2018105026 A1 WO 2018105026A1 JP 2016086133 W JP2016086133 W JP 2016086133W WO 2018105026 A1 WO2018105026 A1 WO 2018105026A1
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unit
image
input
scanning microscope
laser
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French (fr)
Japanese (ja)
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兼太郎 井元
厚志 土井
隆介 田中
秀憲 壷井
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オリンパス株式会社
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Priority to PCT/JP2016/086133 priority Critical patent/WO2018105026A1/en
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

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  • the present invention relates to a laser scanning microscope.
  • a laser beam is scanned on a specimen, fluorescence generated at each scanning position is detected by a photodetector, processing such as integration is performed on the detected fluorescence intensity signal, and the processed fluorescence intensity signal is scanned at a scanning position.
  • a laser scanning microscope that acquires a fluorescence image by associating with information is known (see, for example, Patent Document 1).
  • the present invention has been made in view of the above-described circumstances, and provides a laser scanning microscope that can acquire an image with changed image parameters and feature amounts without irradiating a sample with laser light again.
  • the purpose is that.
  • One embodiment of the present invention includes a scanning unit that scans a sample with laser light, a detection unit that detects signal light generated at each scanning position of the laser light by the scanning unit, and a cycle shorter than an exposure time by the detection unit.
  • the laser scanning microscope includes an image generation unit that generates an image using the detection signal and the scanning position recorded in the recording unit based on image creation information.
  • the scanning unit scans the sample with laser light, the signal light generated at each scanning position of the sample is detected by the detection unit, and the detected detection signal is recorded in the recording unit in association with the scanning position. Is done. Since the detection by the detection unit is performed at a cycle shorter than the exposure time, an image with an exposure time longer than the detection cycle can be generated by the image generation unit. That is, even when a long exposure time is specified in the image creation information first input in the input unit and an image based on the long exposure time is generated, a shorter exposure time is input again in the input unit. By doing so, an image based on a shorter exposure time can be generated. In this case, it is not necessary to re-irradiate the specimen with laser light, and a desired image can be obtained easily and in a short time, and damage to the specimen can be reduced.
  • the detection unit includes a detector that detects the signal light, and an A / D converter that samples the signal detected by the detector and outputs the detection signal.
  • the D converter may perform sampling at a frequency higher than the response speed of the detector. In this way, the signal detected by the detector can be recorded on the recording unit without being missed.
  • the number of pixels may be sufficient as the said image creation information input by the said input part. In this way, even when a small pixel number is specified in the image creation information first input in the input unit and a low-resolution image is generated, a larger pixel number is set in the input unit. By inputting again, a higher resolution image can be generated.
  • the said image creation information input by the said input part may be exposure time.
  • the kind of signal processing performed to the said detection signal may be sufficient as the said image creation information input by the said input part.
  • the signal processing may be noise removal processing. In this way, if sufficient noise removal has not been performed by the noise removal processing that was input first, or if the necessary detection signal has been lost, an image with a modified noise removal processing method Can be regenerated.
  • the signal processing may be threshold processing.
  • the laser light may be an ultrashort pulse laser light
  • the signal light may be fluorescence
  • the signal processing may calculate a fluorescence decay time from the detection signal. In this way, when a fluorescence intensity image is generated by the first input signal processing, a fluorescence lifetime image is generated by selecting a process for calculating the fluorescence decay time as the signal processing. Can be redone.
  • a plurality of the image creation information may be input by the input unit, and the image generation unit may generate a plurality of images based on the input plurality of the image creation information. In this way, a plurality of generated images can be compared.
  • FIG. 2 is a diagram illustrating a case where exposure for three pixels is performed on a horizontal scanning line in the laser scanning microscope of FIG. 1. It is a figure which shows the example of a 3 * 3 pixel fluorescence image acquired by exposure of FIG. It is a figure which shows the case where exposure for 6 pixels is performed on a horizontal scanning line in the laser scanning microscope of FIG. It is a figure which shows the fluorescence image example of 6x6 pixel acquired by exposure of FIG. It is a figure which shows the case where a 3 * 3 pixel fluorescent image is comprised with the exposure time shorter than the exposure time of FIG.
  • a laser scanning microscope 1 includes a scanner (scanning unit) 3 that two-dimensionally scans laser light from a laser light source 2 and a laser scanned by the scanner 3.
  • An objective lens 4 that condenses the fluorescence (signal light) generated in the specimen A while condensing the light on the specimen A is provided.
  • the laser scanning microscope 1 also includes a dichroic mirror 5 that branches the fluorescence collected by the objective lens 4 from the optical path of the laser beam, a detection unit 6 that detects the fluorescence branched by the dichroic mirror 5, and the detection A calculation unit (image generation unit) 7 that generates a fluorescence image from the fluorescence signal detected by the unit 6 is provided. Further, the laser scanning microscope 1 includes a memory (recording unit) 8 that records a fluorescence signal detected by the detection unit 6, an input unit 9 that inputs image creation information, and a control that controls the scanner 3 and the calculation unit 7. And a PC (personal computer) 11 that displays the generated image.
  • Reference numeral 12 in the figure denotes a mirror for forming an optical path.
  • the detection unit 6 is, for example, a PMT (photomultiplier tube, detector) 13 that detects fluorescence and outputs an intensity signal, and an A / D that samples the fluorescence intensity signal output from the PMT 13 and performs A / D conversion.
  • D A / D converter
  • the A / D 14 samples the fluorescence intensity signal output from the PMT 13 at a frequency higher than the response speed of the PMT 13 and outputs a detection signal composed of a digital signal.
  • the memory 8 records the detection signal output from the A / D 14 in association with the scanning position information by the scanner 3 when the detection signal is detected by the PMT 13. Scanning position information by the scanner 3 can be received from the control unit 10. For the scanning position information, the signal output from the scanner 3 may be A / D converted.
  • the input unit 9 allows the operator to input image creation information.
  • Examples of the image creation information include exposure time, the number of pixels, the angle of view, or the type of signal processing.
  • whether or not to recreate an image can be input from the input unit 9.
  • the control unit 10 controls the scanner 3 and controls the calculation unit 7 to generate an image when only the image creation information is input from the input unit 9 without accompanying a re-creation command. Yes. In this case, the control unit 10 controls the swing angle of the scanner 3 according to the information on the angle of view input by the input unit 9.
  • control unit 10 determines the integration time in the calculation unit 7 of the detection signal output from the A / D 14 based on the information on the exposure time and the number of pixels input by the input unit 9. Furthermore, the control unit 10 selects signal processing to be performed by the calculation unit 7 based on the type of signal processing input by the input unit 9.
  • the calculation unit 7 integrates the detection signals from the A / D 14 according to the determined integration time, and uses the control unit 10 as luminance information of each pixel.
  • Image information is generated by associating with the scanning position information sent from 10 and output to the PC 11.
  • the control unit 10 when image creation information is input together with a command for recreating an image at the input unit 9, the control unit 10 is recorded in the memory 8 in the computing unit 7 without controlling the scanner 3. The detected signal is read out, and an image re-creation process is performed in accordance with new image creation information.
  • the control unit 10 controls the scanner 3 to output from the laser light source 2.
  • Laser light is two-dimensionally scanned by the scanner 3 and condensed on the specimen A by the objective lens 4, and fluorescence generated at each scanning position in the specimen A is condensed by the objective lens 4.
  • the fluorescence condensed by the objective lens 4 is branched from the optical path of the laser beam by the dichroic mirror 5, detected by the PMT 13, and sampled by the A / D 14.
  • sampling is performed with a sampling time (frequency) shorter than the response time of the PMT 13, the detection signal by the PMT 13 is acquired without missing.
  • the sampled detection signal is recorded in the memory 8 in association with the scanning position information of the scanner 3 at the time when the fluorescence is detected, and is output to the calculation unit 7.
  • the horizontal scanning lines are divided according to the number of pixels set in the control unit 10 based on the image creation information input in the input unit 9. As an example, when the number of pixels in the horizontal direction is 3, as shown in FIG. 3, the horizontal scanning line is divided into three. In the example shown in FIG. 3, the entire scanning time along each divided scanning line is set as the first exposure time, and an integrated time equal to the first exposure time is set.
  • the luminance value of each pixel is generated by integrating the detection signals input from the A / D 14 for the integration time set in the control unit 10, and at a specific position within the integration time.
  • a fluorescent image of 3 ⁇ 3 pixels is generated and output to the PC 11.
  • the PC 11 performs predetermined image processing on the sent fluorescent image information and displays it.
  • the calculation unit is based on the input image creation information. 7 reads the detection signal from the memory 8 and reconstructs the image information.
  • the calculation unit 7 outputs the detection signal read from the memory 8 to half of the case of 3 ⁇ 3 pixels.
  • a luminance value of each pixel is generated by integration for an integration time equal to the second exposure time, and is associated with scanning position information at a specific position within the integration time.
  • the calculation unit 7 generates a 6 ⁇ 6 pixel fluorescent image and outputs it to the PC 11.
  • the 6 ⁇ when it is desired to create a 6-pixel fluorescence image, the fluorescence image can be acquired without irradiating the specimen A with laser light again. Accordingly, damage to the specimen A due to repeated laser light irradiation can be suppressed, and the specimen A can be maintained in a healthy state for a long time. Further, there is an advantage that it is possible to save time and labor for irradiating the sample A with the laser light and redetecting the fluorescence.
  • the fluorescent image is generated by the first exposure time shown in FIG. 3 and then shown in FIG. As described above, the exposure time alone may be shortened without changing the number of pixels. By doing so, there is an advantage that the fluorescent image can be reconstructed with a shorter exposure time, and the minimum exposure time can be searched.
  • the fluorescence image may be reconstructed by changing the type of signal processing in the calculation unit 7.
  • Examples of signal processing include noise removal processing such as a low-pass filter or threshold processing.
  • a fluorescent image is generated by a detection signal subjected to one type of signal processing
  • different types of signals are detected using the detection signal recorded in the memory 8 without scanning the laser beam again to detect fluorescence. Processing can be performed to reconstruct the fluorescent image.
  • the fluorescence decay time is calculated by using the detection signal recorded in the memory 8.
  • a lifetime image may be generated.
  • the detection signal for only a specific range may be recorded.
  • a range designation unit (not shown) for designating a range of scanning positions to be recorded in the memory 8 may be provided. In this way, it is possible to record only the detection signal in a range where there is a possibility of reconstructing an image with different image creation information, and to reduce the time required for recording and the capacity of the memory 8.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

The purpose of the present invention is to acquire an image wherein the image parameters and feature amounts change without radiating a sample with another laser. A laser scanning microscope (1) according to the present invention comprises: a scanning unit (3) for scanning a sample (A) with laser light; a detection unit (6) for detecting signal light generated at each laser light scanning position of the scanning unit; a recording unit (8) for mapping and recording detection signals detected by the detection unit in a period shorter than an exposure period and each scanning position at a time the detection signal is detected; an input unit (9) for inputting image preparation information; and an image generation unit (7) for generating an image using the detection signals and scanning positions recorded in the recording unit on the basis of the image preparation information entered via the input unit.

Description

レーザ走査型顕微鏡Laser scanning microscope
　本発明は、レーザ走査型顕微鏡に関するものである。 The present invention relates to a laser scanning microscope.
　従来、レーザ光を標本において走査させ、各走査位置において発生する蛍光等を光検出器により検出し、検出された蛍光強度信号に積算等の処理を施すとともに、処理後の蛍光強度信号を走査位置情報と対応づけることにより蛍光画像を取得するレーザ走査型顕微鏡が知られている（例えば、特許文献１参照。）。 Conventionally, a laser beam is scanned on a specimen, fluorescence generated at each scanning position is detected by a photodetector, processing such as integration is performed on the detected fluorescence intensity signal, and the processed fluorescence intensity signal is scanned at a scanning position. A laser scanning microscope that acquires a fluorescence image by associating with information is known (see, for example, Patent Document 1).
国際公開第２０１３－１２２１６９号パンフレットInternational Publication No. 2013-122169 Pamphlet
　しかしながら、光検出器により検出された時系列信号は、画像化により失われるため、画素数や露光時間のような画像パラメータや画像化する特徴量を変更したい場合には、標本に再度レーザ光を照射して蛍光を検出する必要があり、煩雑であるとともに、標本に褪色等のダメージを与える不都合があった。 However, since the time-series signal detected by the photodetector is lost due to imaging, if you want to change image parameters such as the number of pixels or exposure time and the feature quantity to be imaged, laser light is again applied to the specimen. It is necessary to detect fluorescence by irradiation, which is complicated and inconveniently causes damage such as discoloration to the specimen.
　本発明は、上述した事情に鑑みてなされたものであって、レーザ光を標本に再度照射することなく、画像パラメータや特徴量を変更した画像を取得することができるレーザ走査型顕微鏡を提供することを目的としている。 The present invention has been made in view of the above-described circumstances, and provides a laser scanning microscope that can acquire an image with changed image parameters and feature amounts without irradiating a sample with laser light again. The purpose is that.
　本発明の一態様は、標本においてレーザ光を走査させる走査部と、該走査部による前記レーザ光の各走査位置において発生した信号光を検出する検出部と、該検出部により露光時間より短い周期で検出された検出信号と、該検出信号が検出された時点における各前記走査位置とを対応づけて記録する記録部と、画像作成情報を入力する入力部と、該入力部により入力された前記画像作成情報に基づいて、前記記録部に記録されている前記検出信号と前記走査位置とを用いて画像を生成する画像生成部とを備えるレーザ走査型顕微鏡である。 One embodiment of the present invention includes a scanning unit that scans a sample with laser light, a detection unit that detects signal light generated at each scanning position of the laser light by the scanning unit, and a cycle shorter than an exposure time by the detection unit. A recording unit for recording the detection signal detected in step 1 and each scanning position at the time when the detection signal is detected, an input unit for inputting image creation information, and the input unit The laser scanning microscope includes an image generation unit that generates an image using the detection signal and the scanning position recorded in the recording unit based on image creation information.
　本態様によれば、走査部によって標本においてレーザ光を走査させ、標本の各走査位置において発生した信号光が検出部により検出され、検出された検出信号が走査位置と対応づけて記録部に記録される。検出部による検出は、露光時間より短い周期で行われるので、検出周期以上の露光時間による画像を画像生成部において生成することができる。すなわち、入力部において最初に入力された画像作成情報において長い露光時間が指定されていて、長い露光時間に基づいた画像が生成された後であっても、より短い露光時間を入力部において再度入力することにより、より短い露光時間に基づいた画像を生成することができる。この場合に、標本へのレーザ光の再度の照射が不要であり、簡易かつ短時間に所望の画像を得ることができるとともに、標本に与えるダメージを低減することができる。 According to this aspect, the scanning unit scans the sample with laser light, the signal light generated at each scanning position of the sample is detected by the detection unit, and the detected detection signal is recorded in the recording unit in association with the scanning position. Is done. Since the detection by the detection unit is performed at a cycle shorter than the exposure time, an image with an exposure time longer than the detection cycle can be generated by the image generation unit. That is, even when a long exposure time is specified in the image creation information first input in the input unit and an image based on the long exposure time is generated, a shorter exposure time is input again in the input unit. By doing so, an image based on a shorter exposure time can be generated. In this case, it is not necessary to re-irradiate the specimen with laser light, and a desired image can be obtained easily and in a short time, and damage to the specimen can be reduced.
　上記態様においては、前記検出部が、前記信号光を検出する検出器と、該検出器により検出された信号をサンプリングして前記検出信号を出力するＡ／Ｄ変換器とを備え、該Ａ／Ｄ変換器が、前記検出器の応答速度より高い周波数でサンプリングを行ってもよい。
　このようにすることで、検出器により検出された信号を取りこぼしなく記録部に記録することができる。 In the above aspect, the detection unit includes a detector that detects the signal light, and an A / D converter that samples the signal detected by the detector and outputs the detection signal. The D converter may perform sampling at a frequency higher than the response speed of the detector.
In this way, the signal detected by the detector can be recorded on the recording unit without being missed.
　また、上記態様においては、前記入力部により入力される前記画像作成情報が画素数であってもよい。
　このようにすることで、入力部において最初に入力された画像作成情報において小さな画素数が指定されていて、低解像度の画像が生成された後であっても、より大きな画素数を入力部において再度入力することにより、より高解像の画像を生成することができる。 Moreover, in the said aspect, the number of pixels may be sufficient as the said image creation information input by the said input part.
In this way, even when a small pixel number is specified in the image creation information first input in the input unit and a low-resolution image is generated, a larger pixel number is set in the input unit. By inputting again, a higher resolution image can be generated.
　また、上記態様においては、前記入力部により入力される前記画像作成情報が露光時間であってもよい。
　このようにすることで、入力部において最初に入力された画像作成情報において長い露光時間が指定されていて、長い露光時間に基づいた画像が生成された後であっても、より短い露光時間を入力部において再度入力することにより、より短い露光時間に基づいた画像を生成することができる。 Moreover, in the said aspect, the said image creation information input by the said input part may be exposure time.
By doing in this way, even after the long exposure time is specified in the image creation information first input in the input unit and an image based on the long exposure time is generated, a shorter exposure time can be set. By inputting again in the input unit, an image based on a shorter exposure time can be generated.
　また、上記態様においては、前記入力部により入力される前記画像作成情報が、前記検出信号に施す信号処理の種類であってもよい。
　このようにすることで、入力部において最初に入力された画像作成情報において特定種類の信号処理が指定されていて、当該信号処理が施された画像が生成された後であっても、異なる信号処理の種類を入力部において再度入力することにより、異なる信号処理を施した画像を生成することができる。 Moreover, in the said aspect, the kind of signal processing performed to the said detection signal may be sufficient as the said image creation information input by the said input part.
In this way, even after a specific type of signal processing is specified in the image creation information first input by the input unit and an image subjected to the signal processing is generated, a different signal By inputting the type of processing again at the input unit, an image subjected to different signal processing can be generated.
　また、上記態様においては、前記信号処理が、ノイズ除去処理であってもよい。
　このようにすることで、最初に入力されたノイズ除去処理によって、十分なノイズ除去が行われなかった場合、あるいは、必要な検出信号が失われた場合に、ノイズ除去処理の方法を変更した画像を生成し直すことができる。 In the above aspect, the signal processing may be noise removal processing.
In this way, if sufficient noise removal has not been performed by the noise removal processing that was input first, or if the necessary detection signal has been lost, an image with a modified noise removal processing method Can be regenerated.
　また、上記態様においては、前記信号処理が、閾値処理であってもよい。
　このようにすることで、最初に入力された閾値処理によって、必要な検出信号が失われた場合に、閾値を変更して画像を生成し直すことができる。 In the above aspect, the signal processing may be threshold processing.
By doing in this way, when a necessary detection signal is lost by the threshold value process inputted first, it is possible to regenerate an image by changing the threshold value.
　また、前記レーザ光が極短パルスレーザ光であり、前記信号光が蛍光であり、前記信号処理が、前記検出信号から蛍光の減衰時間を算出してもよい。
　このようにすることで、最初に入力された信号処理により、蛍光強度の画像が生成された場合に、信号処理として蛍光の減衰時間を算出する処理を選択することにより、蛍光寿命の画像を生成し直すことができる。 Further, the laser light may be an ultrashort pulse laser light, the signal light may be fluorescence, and the signal processing may calculate a fluorescence decay time from the detection signal.
In this way, when a fluorescence intensity image is generated by the first input signal processing, a fluorescence lifetime image is generated by selecting a process for calculating the fluorescence decay time as the signal processing. Can be redone.
　また、上記態様においては、前記入力部により複数の前記画像作成情報を入力し、前記画像生成部が、入力された複数の前記画像作成情報に基づく複数の画像を生成してもよい。
　このようにすることで、生成された複数の画像を比較することができる。 In the above aspect, a plurality of the image creation information may be input by the input unit, and the image generation unit may generate a plurality of images based on the input plurality of the image creation information.
In this way, a plurality of generated images can be compared.
　また、上記態様においては、前記記録部に記録する前記走査位置の範囲を指定する範囲指定部を備えていてもよい。
　このようにすることで、異なる画像作成情報により画像を生成し直す可能性のある範囲のみの検出信号を記録することができ、記録に要する時間や記録部の容量を削減することができる。 Moreover, in the said aspect, you may provide the range designation | designated part which designates the range of the said scanning position recorded on the said recording part.
In this way, it is possible to record detection signals only in a range where there is a possibility of regenerating an image with different image creation information, and it is possible to reduce the time required for recording and the capacity of the recording unit.
　本発明によれば、レーザ光を標本に再度照射することなく、画像パラメータや特徴量を変更した画像を取得することができるという効果を奏する。 According to the present invention, there is an effect that it is possible to acquire an image in which image parameters and feature amounts are changed without irradiating the sample with laser light again.
本発明の一実施形態に係るレーザ走査型顕微鏡を示すブロック図である。It is a block diagram which shows the laser scanning microscope which concerns on one Embodiment of this invention. 図１のレーザ走査型顕微鏡による輝度情報および位置情報の時間変化を示す図である。It is a figure which shows the time change of the luminance information and position information by the laser scanning microscope of FIG. 図１のレーザ走査型顕微鏡において水平走査線上に３画素分の露光を行う場合を示す図である。FIG. 2 is a diagram illustrating a case where exposure for three pixels is performed on a horizontal scanning line in the laser scanning microscope of FIG. 1. 図３の露光により取得された３×３画素の蛍光画像例を示す図である。It is a figure which shows the example of a 3 * 3 pixel fluorescence image acquired by exposure of FIG. 図１のレーザ走査型顕微鏡において水平走査線上に６画素分の露光を行う場合を示す図である。It is a figure which shows the case where exposure for 6 pixels is performed on a horizontal scanning line in the laser scanning microscope of FIG. 図５の露光により取得された６×６画素の蛍光画像例を示す図である。It is a figure which shows the fluorescence image example of 6x6 pixel acquired by exposure of FIG. 図３の露光時間より短い露光時間により３×３画素の蛍光画像を構成する場合を示す図である。It is a figure which shows the case where a 3 * 3 pixel fluorescent image is comprised with the exposure time shorter than the exposure time of FIG.
　本発明の一実施形態に係るレーザ走査型顕微鏡１について、図面を参照して以下に説明する。
　本実施形態に係るレーザ走査型顕微鏡１は、図１に示されるように、レーザ光源２からのレーザ光を２次元的に走査するスキャナ（走査部）３と、該スキャナ３により走査されたレーザ光を標本Ａに集光する一方、標本Ａにおいて発生した蛍光（信号光）を集光する対物レンズ４とを備えている。 A laser scanning microscope 1 according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a laser scanning microscope 1 according to the present embodiment includes a scanner (scanning unit) 3 that two-dimensionally scans laser light from a laser light source 2 and a laser scanned by the scanner 3. An objective lens 4 that condenses the fluorescence (signal light) generated in the specimen A while condensing the light on the specimen A is provided.
　また、レーザ走査型顕微鏡１は、対物レンズ４により集光された蛍光をレーザ光の光路から分岐するダイクロイックミラー５と、該ダイクロイックミラー５により分岐された蛍光を検出する検出部６と、該検出部６により検出された蛍光信号から蛍光画像を生成する演算部（画像生成部）７とを備えている。さらに、レーザ走査型顕微鏡１は、検出部６により検出された蛍光信号を記録するメモリ（記録部）８と、画像作成情報を入力する入力部９と、スキャナ３および演算部７を制御する制御部１０と、生成された画像を表示するＰＣ（パーソナルコンピュータ）１１とを備えている。図中符号１２は、光路を形成するためのミラーである。 The laser scanning microscope 1 also includes a dichroic mirror 5 that branches the fluorescence collected by the objective lens 4 from the optical path of the laser beam, a detection unit 6 that detects the fluorescence branched by the dichroic mirror 5, and the detection A calculation unit (image generation unit) 7 that generates a fluorescence image from the fluorescence signal detected by the unit 6 is provided. Further, the laser scanning microscope 1 includes a memory (recording unit) 8 that records a fluorescence signal detected by the detection unit 6, an input unit 9 that inputs image creation information, and a control that controls the scanner 3 and the calculation unit 7. And a PC (personal computer) 11 that displays the generated image. Reference numeral 12 in the figure denotes a mirror for forming an optical path.
　検出部６は、例えば、蛍光を検出して強度信号を出力するＰＭＴ（光電子増倍管、検出器）１３と、該ＰＭＴ１３から出力された蛍光強度信号をサンプリングしてＡ／Ｄ変換するＡ／Ｄ（Ａ／Ｄ変換器）１４とを備えている。Ａ／Ｄ１４はＰＭＴ１３から出力された蛍光強度信号を、ＰＭＴ１３の応答速度より高い周波数でサンプリングして、ディジタル信号からなる検出信号を出力するようになっている。 The detection unit 6 is, for example, a PMT (photomultiplier tube, detector) 13 that detects fluorescence and outputs an intensity signal, and an A / D that samples the fluorescence intensity signal output from the PMT 13 and performs A / D conversion. D (A / D converter) 14. The A / D 14 samples the fluorescence intensity signal output from the PMT 13 at a frequency higher than the response speed of the PMT 13 and outputs a detection signal composed of a digital signal.
　メモリ８は、Ａ／Ｄ１４から出力された検出信号と、該検出信号がＰＭＴ１３により検出された時点におけるスキャナ３による走査位置情報とを対応づけて記録するようになっている。スキャナ３による走査位置情報は、制御部１０から受け取ることができる。走査位置情報は、スキャナ３から出力された信号をＡ／Ｄ変換してもよい。 The memory 8 records the detection signal output from the A / D 14 in association with the scanning position information by the scanner 3 when the detection signal is detected by the PMT 13. Scanning position information by the scanner 3 can be received from the control unit 10. For the scanning position information, the signal output from the scanner 3 may be A / D converted.
　入力部９は、操作者が画像作成情報を入力するようになっている。画像作成情報としては、例えば、露光時間、画素数、画角あるいは信号処理の種別等を挙げることができる。また、入力部９からは、画像の再作成の有無を入力することができるようになっている。 The input unit 9 allows the operator to input image creation information. Examples of the image creation information include exposure time, the number of pixels, the angle of view, or the type of signal processing. In addition, whether or not to recreate an image can be input from the input unit 9.
　制御部１０は、入力部９から再作成の指令を伴うことなく、画像作成情報のみが入力されたときには、スキャナ３を制御するとともに、演算部７を制御して画像を生成させるようになっている。この場合には、制御部１０は、入力部９により入力された画角の情報に従って、スキャナ３の振り角を制御するようになっている。 The control unit 10 controls the scanner 3 and controls the calculation unit 7 to generate an image when only the image creation information is input from the input unit 9 without accompanying a re-creation command. Yes. In this case, the control unit 10 controls the swing angle of the scanner 3 according to the information on the angle of view input by the input unit 9.
　また、制御部１０は、入力部９により入力された露光時間および画素数の情報に基づいて、Ａ／Ｄ１４から出力される検出信号の演算部７における積算時間を決定するようになっている。
　さらに、制御部１０は、入力部９により入力された信号処理の種別に基づいて、演算部７において実施させる信号処理を選択するようになっている。 Further, the control unit 10 determines the integration time in the calculation unit 7 of the detection signal output from the A / D 14 based on the information on the exposure time and the number of pixels input by the input unit 9.
Furthermore, the control unit 10 selects signal processing to be performed by the calculation unit 7 based on the type of signal processing input by the input unit 9.
　演算部７は、制御部１０において、画素数に応じた積算時間が決定されたときには、決定された積算時間に従って、Ａ／Ｄ１４からの検出信号を積算し、各画素の輝度情報として、制御部１０から送られてきた走査位置情報に対応づけることにより、画像情報を生成し、ＰＣ１１に出力するようになっている。 When the control unit 10 determines the integration time according to the number of pixels, the calculation unit 7 integrates the detection signals from the A / D 14 according to the determined integration time, and uses the control unit 10 as luminance information of each pixel. Image information is generated by associating with the scanning position information sent from 10 and output to the PC 11.
　また、本実施形態においては、入力部９において画像を再作成の指令とともに画像作成情報が入力されたときには、制御部１０はスキャナ３を制御することなく、演算部７にメモリ８内に記録されている検出信号を読み出させ、新たな画像作成情報に応じて画像の再作成処理を施させるようになっている。 Further, in the present embodiment, when image creation information is input together with a command for recreating an image at the input unit 9, the control unit 10 is recorded in the memory 8 in the computing unit 7 without controlling the scanner 3. The detected signal is read out, and an image re-creation process is performed in accordance with new image creation information.
　このように構成された本実施形態に係るレーザ走査型顕微鏡１の作用について、以下に説明する。
　本実施形態に係るレーザ走査型顕微鏡１によれば、入力部９から再作成指令を伴うことなく画像作成情報が入力されると、制御部１０がスキャナ３を制御して、レーザ光源２からのレーザ光がスキャナ３において２次元的に走査され、対物レンズ４によって標本Ａに集光され、標本Ａにおける各走査位置において発生した蛍光が対物レンズ４により集光される。 The operation of the laser scanning microscope 1 according to the present embodiment configured as described above will be described below.
According to the laser scanning microscope 1 according to the present embodiment, when image creation information is input from the input unit 9 without a re-creation command, the control unit 10 controls the scanner 3 to output from the laser light source 2. Laser light is two-dimensionally scanned by the scanner 3 and condensed on the specimen A by the objective lens 4, and fluorescence generated at each scanning position in the specimen A is condensed by the objective lens 4.
　対物レンズ４により集光された蛍光はダイクロイックミラー５によってレーザ光の光路から分岐され、ＰＭＴ１３によって検出されて、Ａ／Ｄ１４によりサンプリングされる。本実施形態においては、図２に示されるように、ＰＭＴ１３の応答時間より短いサンプリング時間（周波数）でサンプリングされるので、ＰＭＴ１３による検出信号が取りこぼしなく取得される。 The fluorescence condensed by the objective lens 4 is branched from the optical path of the laser beam by the dichroic mirror 5, detected by the PMT 13, and sampled by the A / D 14. In the present embodiment, as shown in FIG. 2, since sampling is performed with a sampling time (frequency) shorter than the response time of the PMT 13, the detection signal by the PMT 13 is acquired without missing.
　そして、サンプリングされた検出信号は、当該蛍光が検出された時点におけるスキャナ３の走査位置情報と対応づけてメモリ８に記録されるとともに、演算部７に出力される。
　演算部７においては、入力部９において入力された画像作成情報に基づいて制御部１０において設定された画素数に応じて、水平方向の走査線が分割される。例として、水平方向の画素数が３である場合、図３に示されるように、水平方向の走査線が３分割される。図３に示す例では、分割された各走査線に沿う走査時間全体が第１露光時間として設定され、該第１露光時間に等しい積算時間が設定されている。 The sampled detection signal is recorded in the memory 8 in association with the scanning position information of the scanner 3 at the time when the fluorescence is detected, and is output to the calculation unit 7.
In the calculation unit 7, the horizontal scanning lines are divided according to the number of pixels set in the control unit 10 based on the image creation information input in the input unit 9. As an example, when the number of pixels in the horizontal direction is 3, as shown in FIG. 3, the horizontal scanning line is divided into three. In the example shown in FIG. 3, the entire scanning time along each divided scanning line is set as the first exposure time, and an integrated time equal to the first exposure time is set.
　演算部７においては、制御部１０において設定された積算時間だけＡ／Ｄ１４から入力されてきた検出信号が積算されることにより、各画素の輝度値が生成され、当該積算時間内の特定位置における走査位置情報と対応づけられることにより、図４に示されるように、３×３画素の蛍光画像が生成され、ＰＣ１１に出力される。
　ＰＣ１１は、送られてきた蛍光画像情報に所定の画像処理を施して表示する。 In the calculation unit 7, the luminance value of each pixel is generated by integrating the detection signals input from the A / D 14 for the integration time set in the control unit 10, and at a specific position within the integration time. By associating with the scanning position information, as shown in FIG. 4, a fluorescent image of 3 × 3 pixels is generated and output to the PC 11.
The PC 11 performs predetermined image processing on the sent fluorescent image information and displays it.
　一方、ＰＣ１１により表示された蛍光画像を確認した操作者が、入力部９から蛍光画像の再作成指令を伴って画像作成情報を入力した場合には、入力された画像作成情報に基づいて演算部７がメモリ８から検出信号を読み出し、画像情報を再構成する。 On the other hand, when an operator who has confirmed the fluorescence image displayed by the PC 11 inputs image creation information from the input unit 9 with a fluorescence image re-creation command, the calculation unit is based on the input image creation information. 7 reads the detection signal from the memory 8 and reconstructs the image information.
　例えば、操作者が、６×６画素の画素数を指定した場合には、演算部７は、図５に示されるように、メモリ８から読み出した検出信号を３×３画素の場合の半分の第２露光時間に等しい積算時間だけ積算して各画素の輝度値を生成し、当該積算時間内の特定位置における走査位置情報と対応づける。これにより、演算部７において、図６に示されるように、６×６画素の蛍光画像が生成され、ＰＣ１１に出力される。 For example, when the operator specifies the number of pixels of 6 × 6 pixels, as shown in FIG. 5, the calculation unit 7 outputs the detection signal read from the memory 8 to half of the case of 3 × 3 pixels. A luminance value of each pixel is generated by integration for an integration time equal to the second exposure time, and is associated with scanning position information at a specific position within the integration time. As a result, as shown in FIG. 6, the calculation unit 7 generates a 6 × 6 pixel fluorescent image and outputs it to the PC 11.
　この場合において、本実施形態に係るレーザ走査型顕微鏡１によれば、レーザ光を標本Ａに照射して取得された検出信号に基づいて３×３画素の蛍光画像を生成された後に、６×６画素の蛍光画像の作成を望む場合に、レーザ光を再度標本Ａに照射することなく蛍光画像を取得することができる。
　したがって、度重なるレーザ光の照射により標本Ａに与えるダメージを抑えることができ、長時間にわたり標本Ａを健全な状態に維持することができる。
　また、レーザ光を標本Ａに照射して蛍光を再検出する手間と時間を省くことができるという利点がある。 In this case, according to the laser scanning microscope 1 according to the present embodiment, after the 3 × 3 pixel fluorescent image is generated based on the detection signal obtained by irradiating the sample A with the laser light, the 6 × When it is desired to create a 6-pixel fluorescence image, the fluorescence image can be acquired without irradiating the specimen A with laser light again.
Accordingly, damage to the specimen A due to repeated laser light irradiation can be suppressed, and the specimen A can be maintained in a healthy state for a long time.
Further, there is an advantage that it is possible to save time and labor for irradiating the sample A with the laser light and redetecting the fluorescence.
　なお、本実施形態においては、画素数および露光時間を同時に変化させる場合について説明したが、これに代えて、図３に示される第１露光時間により蛍光画像を生成した後に、図７に示されるように、画素数を変化させることなく露光時間のみを短縮する方向に変化させることにしてもよい。
　このようにすることで、より短い露光時間で蛍光画像を再構成することができ、必要最小限の露光時間を探索することができるという利点がある。 In the present embodiment, the case where the number of pixels and the exposure time are changed at the same time has been described. Instead, the fluorescent image is generated by the first exposure time shown in FIG. 3 and then shown in FIG. As described above, the exposure time alone may be shortened without changing the number of pixels.
By doing so, there is an advantage that the fluorescent image can be reconstructed with a shorter exposure time, and the minimum exposure time can be searched.
　また、本実施形態においては、演算部７における信号処理の種別を変更して蛍光画像を再構成することにしてもよい。信号処理としては、例えば、ローパスフィルタあるいは閾値処理等のノイズ除去処理を挙げることができる。 Further, in the present embodiment, the fluorescence image may be reconstructed by changing the type of signal processing in the calculation unit 7. Examples of signal processing include noise removal processing such as a low-pass filter or threshold processing.
　一の種類の信号処理を施した検出信号により蛍光画像を生成した後に、再度レーザ光を走査して蛍光を検出することなく、メモリ８に記録しておいた検出信号を用いて異なる種類の信号処理を施して蛍光画像を再構成することができる。 After a fluorescent image is generated by a detection signal subjected to one type of signal processing, different types of signals are detected using the detection signal recorded in the memory 8 without scanning the laser beam again to detect fluorescence. Processing can be performed to reconstruct the fluorescent image.
　また、レーザ走査型顕微鏡１として、極短パルスレーザ光を照射する多光子励起型顕微鏡を用いる場合には、メモリ８に記録しておいた検出信号を用いて蛍光の減衰時間を算出して蛍光寿命画像を生成することにしてもよい。また、蛍光画像と蛍光寿命画像とを同時に表示することにしてもよい。これにより、取得された検出信号に基づいて異なる種類の特徴量を表す画像を構成することができるという利点がある。 When a multi-photon excitation type microscope that irradiates an ultrashort pulse laser beam is used as the laser scanning microscope 1, the fluorescence decay time is calculated by using the detection signal recorded in the memory 8. A lifetime image may be generated. Moreover, you may decide to display a fluorescence image and a fluorescence lifetime image simultaneously. Accordingly, there is an advantage that images representing different types of feature amounts can be configured based on the acquired detection signals.
　また、本実施形態においては、蛍光画像が取得された後に入力部９から単一の画像作成情報を入力する場合について説明したが、これに代えて、蛍光画像の取得前に複数の画像作成情報を入力しておき、メモリ８に記録された検出信号から異なる画像作成情報に基づく異なる画像を生成し、ＰＣ１１に表示することによって、操作者に選択させることにしてもよい。 In the present embodiment, the case where single image creation information is input from the input unit 9 after the fluorescence image is acquired has been described. Instead, a plurality of pieces of image creation information are acquired before the fluorescence image is acquired. , And a different image based on different image creation information may be generated from the detection signal recorded in the memory 8 and displayed on the PC 11 to allow the operator to select it.
　また、本実施形態においては、走査範囲の全体について検出信号をメモリ８に記録することに代えて、特定の範囲のみの検出信号を記録することにしてもよい。この場合には、メモリ８に記録する走査位置の範囲を指定する範囲指定部（図示略）を備えていてもよい。
　このようにすることで、異なる画像作成情報により画像を再構成する可能性のある範囲のみの検出信号を記録して、記録に要する時間やメモリ８の容量を削減することができる。 In the present embodiment, instead of recording the detection signal in the memory 8 for the entire scanning range, the detection signal for only a specific range may be recorded. In this case, a range designation unit (not shown) for designating a range of scanning positions to be recorded in the memory 8 may be provided.
In this way, it is possible to record only the detection signal in a range where there is a possibility of reconstructing an image with different image creation information, and to reduce the time required for recording and the capacity of the memory 8.
　１　レーザ走査型顕微鏡
　３　スキャナ（走査部）
　６　検出部
　７　演算部（画像生成部）
　８　メモリ（記録部）
　９　入力部
　１３　ＰＭＴ（検出器）
　１４　Ａ／Ｄ（Ａ／Ｄ変換器）
　Ａ　標本 1 Laser scanning microscope 3 Scanner (scanning unit)
6 detection unit 7 calculation unit (image generation unit)
8 memory (recording part)
9 Input 13 PMT (detector)
14 A / D (A / D converter)
A specimen

Claims (10)

  1. 　標本においてレーザ光を走査させる走査部と、
    　該走査部による前記レーザ光の各走査位置において発生した信号光を検出する検出部と、
    　該検出部により露光時間より短い周期で検出された検出信号と、該検出信号が検出された時点における各前記走査位置とを対応づけて記録する記録部と、
    　画像作成情報を入力する入力部と、
    　該入力部により入力された前記画像作成情報に基づいて、前記記録部に記録されている前記検出信号と前記走査位置とを用いて画像を生成する画像生成部とを備えるレーザ走査型顕微鏡。     A scanning section for scanning the sample with laser light;
    A detection unit for detecting signal light generated at each scanning position of the laser beam by the scanning unit;
    A recording unit that records a detection signal detected by the detection unit in a cycle shorter than an exposure time and each scanning position at a time when the detection signal is detected;
    An input unit for inputting image creation information;
    A laser scanning microscope comprising: an image generation unit configured to generate an image using the detection signal recorded in the recording unit and the scanning position based on the image creation information input by the input unit.
  2. 　前記検出部が、前記信号光を検出する検出器と、該検出器により検出された信号をサンプリングして前記検出信号を出力するＡ／Ｄ変換器とを備え、
    　該Ａ／Ｄ変換器が、前記検出器の応答速度より高い周波数でサンプリングを行う請求項１に記載のレーザ走査型顕微鏡。     The detection unit includes a detector that detects the signal light, and an A / D converter that samples the signal detected by the detector and outputs the detection signal,
    The laser scanning microscope according to claim 1, wherein the A / D converter performs sampling at a frequency higher than a response speed of the detector.
  3. 　前記入力部により入力される前記画像作成情報が画素数である請求項１または請求項２に記載のレーザ走査型顕微鏡。 The laser scanning microscope according to claim 1 or 2, wherein the image creation information input by the input unit is the number of pixels.
  4. 　前記入力部により入力される前記画像作成情報が露光時間である請求項１または請求項２に記載のレーザ走査型顕微鏡。 3. The laser scanning microscope according to claim 1, wherein the image creation information input by the input unit is an exposure time.
  5. 　前記入力部により入力される前記画像作成情報が、前記検出信号に施す信号処理の種類である請求項１または請求項２に記載のレーザ走査型顕微鏡。 The laser scanning microscope according to claim 1 or 2, wherein the image creation information input by the input unit is a type of signal processing applied to the detection signal.
  6. 　前記信号処理が、ノイズ除去処理である請求項５に記載のレーザ走査型顕微鏡。 6. The laser scanning microscope according to claim 5, wherein the signal processing is noise removal processing.
  7. 　前記信号処理が、閾値処理である請求項５に記載のレーザ走査型顕微鏡。 The laser scanning microscope according to claim 5, wherein the signal processing is threshold processing.
  8. 　前記レーザ光が極短パルスレーザ光であり、
    　前記信号光が蛍光であり、
    　前記信号処理が、前記検出信号から蛍光の減衰時間を算出する請求項５に記載のレーザ走査型顕微鏡。     The laser beam is an ultrashort pulse laser beam,
    The signal light is fluorescent;
    The laser scanning microscope according to claim 5, wherein the signal processing calculates a fluorescence decay time from the detection signal.
  9. 　前記入力部により複数の前記画像作成情報を入力し、
    　前記画像生成部が、入力された複数の前記画像作成情報に基づく複数の画像を生成する請求項１から請求項８のいずれかに記載のレーザ走査型顕微鏡。     Input a plurality of the image creation information by the input unit,
    9. The laser scanning microscope according to claim 1, wherein the image generation unit generates a plurality of images based on the plurality of input image creation information.
  10. 　前記記録部に記録する前記走査位置の範囲を指定する範囲指定部を備える請求項１から請求項９のいずれかに記載のレーザ走査型顕微鏡。
     
     
          The laser scanning microscope according to any one of claims 1 to 9, further comprising a range designating unit that designates a range of the scanning position to be recorded in the recording unit.


PCT/JP2016/086133 2016-12-06 2016-12-06 Laser scanning microscope WO2018105026A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008242227A (en) * 2007-03-28 2008-10-09 Olympus Corp Scanning microscope and adjustment method for the same
JP2014153556A (en) * 2013-02-08 2014-08-25 Olympus Corp Laser microscope apparatus
JP2014158253A (en) * 2013-01-18 2014-08-28 Olympus Corp Light detection circuit and microscope system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008242227A (en) * 2007-03-28 2008-10-09 Olympus Corp Scanning microscope and adjustment method for the same
JP2014158253A (en) * 2013-01-18 2014-08-28 Olympus Corp Light detection circuit and microscope system
JP2014153556A (en) * 2013-02-08 2014-08-25 Olympus Corp Laser microscope apparatus

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