JP6016495B2 - Scanning microscope - Google Patents

Scanning microscope Download PDF

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JP6016495B2
JP6016495B2 JP2012157449A JP2012157449A JP6016495B2 JP 6016495 B2 JP6016495 B2 JP 6016495B2 JP 2012157449 A JP2012157449 A JP 2012157449A JP 2012157449 A JP2012157449 A JP 2012157449A JP 6016495 B2 JP6016495 B2 JP 6016495B2
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image
scale pattern
scanning
illumination light
observation object
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JP2014021184A (en
Inventor
誠 橋爪
誠 橋爪
文紀 兵藤
文紀 兵藤
廉士 澤田
廉士 澤田
晃 森田
晃 森田
河村 幸則
幸則 河村
鈴木 健
健 鈴木
範明 石河
範明 石河
工藤 高裕
高裕 工藤
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国立大学法人九州大学
富士電機株式会社
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Description

  The present invention relates to a scanning microscope.

  As a scanning microscope, for example, in a confocal microscope, light such as a laser emitted from a light source is collected by an objective lens and focused on the surface of an observation object. The focused light becomes reflected light from the observation object, and only the light selected by the slit (pinhole) is transmitted to the detector. A two-dimensional image can be acquired by moving light from the light source in a two-dimensional direction on the surface of the observation object by the scanning means.

  As scanning means for such a scanning microscope, Patent Document 1 discloses that a piezoelectric sensor is used to scan the tip of an optical fiber as a light source by electromagnetic induction resonance vibration and to synchronize this vibration with mechanical scanning with certainty. Describes a combination of electromagnetically induced resonance vibration and hydraulic motion for position feedback.

  Patent Document 2 discloses a method using an electromagnet as a scanning method of an optical fiber which is a light source, and a method using a sensor coil for vibration detection.

JP-A-3-87804 JP 2008-116922 A

  As described above, when a device such as a piezoelectric sensor or a sensor coil is used as the scanning means of the scanning microscope, parts are added for this purpose, and there is a problem that the scanning microscope becomes large and costs increase.

  SUMMARY OF THE INVENTION An object of the present invention is to provide a scanning microscope that can control scanning means without using a special device, and can achieve downsizing and cost reduction.

  The present invention is a scanning microscope, and is disposed between a light source for illuminating illumination light, an objective lens for condensing the illumination light on the surface of the observation object, and the objective lens and the observation object. A transmission plate that transmits the illumination light, the transmission plate having a scale pattern on the surface thereof, a detector that detects reflected light from the observation object, and the illumination light on the surface of the observation object And an image processing apparatus that generates an image from information from the detector and the scanning unit, and based on the image of the scale pattern in the generated image, the illumination light of the scanning unit And an image processing device for controlling the movement of the image.

  It is preferable that the scale pattern is a pattern parallel or orthogonal to a scanning axis that is a moving direction of the illumination light of the scanning unit. Moreover, it is preferable that the said scale pattern is a pattern which avoided the center part of the visual field in which the said observation target object is observed in the said permeation | transmission board.

  It is preferable that the image processing device obtains a median value of a grayscale distribution of the scale pattern image in the generated image.

  According to the present invention, by arranging the transmission plate having the scale pattern between the observation object and the objective lens as described above, based on the size of the image of the scale pattern generated in the generated image, the scanning unit Therefore, the movement of the illumination light of the scanning means can be controlled. Therefore, it is possible to control the scanning means without using special equipment as in the prior art, and it is possible to reduce the size and cost of the scanning microscope.

1 is a cross-sectional view schematically showing an embodiment of a scanning microscope according to the present invention. It is a top view which shows the permeation | transmission surface of the glass window of the scanning microscope shown in FIG. It is a figure which shows the example of imaging of the scale pattern shown in FIG. It is a graph which shows the relationship between the scale pattern image shown in FIG. 3, and an amplitude amount. It is a top view which shows the other example of the scale pattern drawn on the glass window. FIG. 3 is a diagram showing a density distribution of a scale pattern image in the imaging of the scale pattern shown in FIG. 2.

  Hereinafter, an embodiment of a scanning microscope according to the present invention will be described with reference to the accompanying drawings. In this embodiment, a confocal microscope is used as an example of a scanning microscope. However, it will be understood from the following description that the present invention can be applied to other types of scanning microscopes.

  As shown in FIG. 1, a confocal microscope 100 includes a light source 1 that irradiates illumination light 20, a semitransparent mirror 3 that deflects illumination light from the light source, and condenses the deflected illumination light on an observation object 2. Objective lens 5, eyepiece 4 that collects reflected light 20 s emitted from the observation object, and detector 8 that is disposed at the focal position of eyepiece 4. Further, the confocal microscope 100 includes a control device 15 that controls the vibration of the semitransparent mirror 3 and an image processing device 9 that creates a two-dimensional image based on information from the control device 15 and the detector 8.

  As the light source 1, for example, a laser light source, a mercury lamp, a halogen lamp, or the like can be used. The translucent mirror 3 is a beam splitter that reflects the illumination light 20 emitted from the light source 1 and transmits the reflected light 20s from the observation object.

  A mirror axis 7 that vibrates the semitransparent mirror 3 is disposed on a central axis 21 that passes through the centers of the eyepiece lens 4 and the objective lens 5. The semi-transparent mirror 3 is based on the mirror axis 7 so that the focal point 2s of the irradiated light scans the surface of the observation object 2 two-dimensionally (that is, moves in two directions, the X-axis direction and the Y-axis direction). It is fixed so that it can vibrate. For example, when the vibration is performed so that the angle θ between the surface of the semitransparent mirror 3 and the central axis 21 changes, the focal point 2s on the surface of the observation object 2 moves in the Y-axis direction.

  The confocal microscope 100 includes a glass window 10 disposed so as to be positioned between the observation object 2 and the objective lens 5. As long as the glass window 10 transmits the illumination light 20, the material is not particularly limited to glass. As shown in FIG. 2, the glass window 10 has a scale pattern 11 on its surface. The scale pattern 11 is a square pattern in the present embodiment. That is, the scale pattern 11 is formed in the glass window 10 so as to be parallel and orthogonal to the X axis and Y axis, which are scanning axes, and to avoid the central portion of the observable visual field region.

  Between the eyepiece 4 and the detector 8, a slit plate 12 having a slit 12 s at the center is arranged so that the plate surface is orthogonal to the central axis 21.

  The control device 15 is electrically connected to control the vibration of the translucent mirror 3 and to send a signal of the vibration information to the image processing device 9. Further, the detector 8 is electrically connected so as to send the information of the detected reflected light 20 s to the image processing device 9.

  According to the confocal microscope 100 having such a configuration, first, the irradiation light 20 is emitted from the light source 1 as shown in FIG. The illumination light 20 is deflected by the translucent mirror 3 and travels toward the objective lens 5 located on the center line 21. The illumination light 20 is refracted and condensed by the objective lens 5, passes through the glass window 10, and forms a focal point 2 s on the surface of the observation object 2.

  The focused illumination light 20 then becomes reflected light 20s from the observation object 2, passes through the glass window 10 and the objective lens 5 through a path opposite to the path of the illumination light 20, and further translucent mirror 3 Transparent. The reflected light 20 s transmitted through the semitransparent mirror 3 is refracted by the eyepiece lens 4, and only the light selected by the slit 12 s of the slit plate 12 is detected by the detector 8. The reflected light 20 s is converted into an electrical signal by the detector 8 and sent to the image processing device 9.

  In order to perform two-dimensional scanning, the translucent mirror 3 is vibrated with the mirror axis 7 as a base point. Thereby, the focal point 2s of the illumination light 20 moves on the surface of the observation object 2 in the X-axis direction and the Y-axis direction, so that the reflected light 20s can be detected by the detector 8. The vibration signal of the translucent mirror 3 is sent from the image processing device 9 to the control device 15. Therefore, the image processing apparatus 9 can create a two-dimensional image of the observation object by synchronizing with the vibration signal.

  In this two-dimensional image, the scale pattern 11 is acquired in a slightly blurred form. For example, as shown in FIG. 3, the scale pattern 11 is visualized as a scale pattern image 11a that appears large in the acquired image 30, a scale pattern image 11c that appears small, an intermediate scale pattern image 11c, or the like. Alternatively, the scale pattern 11 may not be imaged at all on the acquired image 30.

  This is due to the difference in the vibration width of the translucent mirror 3. When the width of vibration is very small, the illumination light 20 moves only within the scale pattern 11, so that no image of the scale pattern is formed in the acquired image. When the width of vibration increases and the illumination light 20 moves outside the frame of the scale pattern 11, an image of the scale pattern is formed in the acquired image. The larger the vibration width, the larger the illumination light 20 moves out of the frame of the scale pattern 11, and the smaller the scale pattern image formed in the acquired image 30.

  This relationship is shown in FIG. As shown in FIG. 4, as the vibration width (amplitude) A increases as Aa, Ab, and Ac, the size of the scale pattern image in the acquired image decreases as 11a, 11b, and 11c. That is, the amplitude amount of the translucent mirror 3 can be obtained from the size of the scale pattern image in the acquired image. The size of the desired scale pattern image is caused by the size of the scale pattern 11 drawn on the glass window 10. For example, the amplitude of the semitransparent mirror 3 is controlled so as to have a predetermined size as in the scale pattern image 11b.

  The scale pattern 11 drawn on the glass window 10 is not limited to the pattern shown in FIG. 2, and may be a pattern shown in FIG. 5, for example. In addition to a pattern parallel to and perpendicular to the X axis and Y axis, which are scanning axes, a memory pattern can be added to the X axis and the Y axis, and a concentric pattern smaller than the outer edge of the glass window 10 can be added. .

  The scale pattern 11 is preferably a pattern that is parallel and orthogonal to the X axis and Y axis, which are scanning axes. However, even if the scale pattern 11 is not a pattern that is parallel or orthogonal, the scale pattern image in the acquired image is the X axis or Y axis. By rotating the acquired image so as to be parallel or orthogonal to the axis, the amplitude amount of the semitransparent mirror 3 can be similarly controlled.

  In this way, by arranging the glass window 10 having the scale pattern 11 between the observation object 2 and the objective lens 5, the width of vibration of the translucent mirror 3 can be controlled. Therefore, it is possible to reduce the size and cost of the scanning microscope without using special equipment.

  Further, since the scale pattern 11 of the glass window 10 is located closer to the objective lens 5 than the surface of the observation object 2 where the focal point 2s is located, in the acquired image, it becomes a defocused image without being focused. For example, as shown in FIG. 6, even if the scale pattern 11 of the glass window 10 is a thin line, the scale pattern image obtained in the line S has a spread with the center as the maximum density. Therefore, the image processing device 9 can calculate the density distribution 11t of the obtained scale pattern image and obtain the center of the density distribution. The center position of this density distribution is the position of the scale pattern 11. Thus, even if the scale pattern 11 is not focused, the exact position of the scale pattern 11 can be obtained from the scale pattern image, and thus the amplitude amount of the translucent mirror 3 can be obtained accurately.

In the above embodiment, the scale pattern 11 is provided in the glass window 10 positioned between the observation object 2 and the objective lens 5. However, the present invention is not limited to this, and the image processing apparatus 9 is used. Any place where a scale pattern image is formed on the obtained two- dimensional image may be used. For example, in the case of the confocal microscope 100 shown in FIG. Since the size of the obtained scale pattern image differs, the amplitude amount of the semitransparent mirror 3 can be controlled similarly.

DESCRIPTION OF SYMBOLS 1 Light source 2 Observation object 2s Focus 3 Translucent mirror 4 Eyepiece 5 Objective lens 7 Mirror axis 8 Detector 9 Image processing apparatus 10 Glass window 11 Scale pattern 12 Slit plate 12s Slit 15 Controller 20 Light 20s Reflected light

Claims (4)

  1. A light source that emits illumination light;
    Scanning means for moving the illumination light on the surface of the observation object;
    A transmission plate disposed between the scanning means and the observation object and transmitting the illumination light, the transmission plate having a scale pattern on its surface;
    A detector for detecting reflected light from the observation object;
    An image processing apparatus that generates an image from information from the detector and the scanning unit, the movement of the illumination light of the scanning unit based on an image of the scale pattern in the image generated by the image processing apparatus A scanning microscope comprising: an image processing device for controlling the image.
  2.   The scanning microscope according to claim 1, wherein the scale pattern is a pattern parallel or orthogonal to a scanning axis that is a moving direction of the illumination light of the scanning unit.
  3.   The scanning microscope according to claim 1, wherein the scale pattern is a pattern that avoids a central portion of a visual field in which the observation object is observed on the transmission plate.
  4. The image processing apparatus, the image of the scale pattern in an image that the generated scanning microscope according to claim 1 for determining the center position of the shade distribution of the image.
JP2012157449A 2012-07-13 2012-07-13 Scanning microscope Active JP6016495B2 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101823253B1 (en) * 2016-07-29 2018-03-14 장현식 Individual ports and strawberry seedling cultivation method using the same goseol formal place for a strawberry nursery place.

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09197280A (en) * 1996-01-19 1997-07-31 Olympus Optical Co Ltd Scanning linearlity correcting method for scanning type optical microscope
EP0978009B1 (en) * 1998-02-20 2009-05-13 Leica Microsystems CMS GmbH System for calibrating a laser scanning microscope
JP4819991B2 (en) * 2000-09-20 2011-11-24 オリンパス株式会社 Method and apparatus for adjusting magnification of scanning optical microscope
JP2002098901A (en) * 2000-09-22 2002-04-05 Olympus Optical Co Ltd Scanning laser microscope
US20060256342A1 (en) * 2003-03-28 2006-11-16 Wong Lid B Confocal microscope system for real-time simultaneous temporal measurements of metachronal wave period and ciliary beat frequency
JP5378187B2 (en) * 2009-12-09 2013-12-25 オリンパス株式会社 Scanning microscope
JP2011186060A (en) * 2010-03-05 2011-09-22 Nikon Corp Laser scanning-type microscope and control method

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101823253B1 (en) * 2016-07-29 2018-03-14 장현식 Individual ports and strawberry seedling cultivation method using the same goseol formal place for a strawberry nursery place.

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