JP4276974B2 - Laser scanning observation device - Google Patents

Description

The present invention relates to a laser scanning observation apparatus .

Conventionally, as a device for observing the function of cells, etc. by irradiating a sample such as a living body with excitation light from its surface and detecting fluorescence emitted from a relatively deep position below the surface of the sample, a multiphoton excitation type Is known (for example, refer to Patent Document 1).
This multiphoton excitation type measuring apparatus is configured to detect fluorescence emitted from a sample by an external photomultiplier tube connected by a single mode fiber.
JP 2002-243641 A (the third page etc.)

  However, the single mode fiber has a disadvantage that the core diameter is thin and the fluorescence returning from the sample is limited, so that it cannot be measured efficiently. For the purpose of measuring fluorescence efficiently, the photomultiplier tube is preferably arranged immediately after the objective lens of the measurement head. However, since the photomultiplier tube is relatively large, the measurement head becomes larger. Therefore, it is not suitable for use in various postures and positions according to the site to be observed of the sample, for example, when observing experimental small animals alive (in vivo).

  On the other hand, it is conceivable to use a multimode fiber as an optical fiber that has a large core diameter and does not restrict fluorescence from the sample. However, since a multimode fiber propagates light in many transmission modes, it is extremely short from a laser light source. When a pulse laser is incident, multimode dispersion occurs and the pulse width becomes long. If it is used as it is, there is a disadvantage that the multiphoton excitation effect cannot be generated efficiently.

  The present invention has been made in view of the above-mentioned circumstances, and reduces the loss of the amount of fluorescence obtained from a sample while irradiating the sample with an extremely short pulse laser with a short pulse width emitted from a laser light source. An object of the present invention is to provide a laser scanning observation apparatus capable of obtaining a fluorescent image and capable of configuring a measurement head compactly.

In order to achieve the above object, the present invention provides the following means.
In the present invention, an optical unit including a laser light source that emits an ultrashort pulse laser beam, an imaging unit that detects return light from the sample, and optical scanning that scans the sample with the ultrashort pulse laser beam from the laser light source. And a measurement head comprising an objective optical system that forms an image of an ultrashort pulse laser beam scanned by the optical scanning unit on a sample, a multimode fiber connecting the optical unit and the measurement head, and the measurement And a pinhole means made of a material that transmits visible light and blocks ultrashort pulse laser light, and the pinhole means is formed by a coating applied to an end face of the multimode fiber. A laser scanning observation apparatus is provided.

  According to the present invention, the ultrashort pulse laser beam emitted from the laser light source propagates through the multimode fiber and enters the measurement head, and is transmitted through the pinhole means in the measurement head. Since the pinhole means is made of a material that blocks the ultrashort pulse laser light, only the light near the center of the fiber core among the ultrashort pulse laser light that has passed through the multimode fiber is allowed to pass through the pinhole means.

  In the vicinity of the center of the fiber core of the multimode fiber, only the lowest-order mode ultrashort pulse laser beam is emitted, and therefore, only the lowest-order mode ultrashort pulse laser beam is allowed to pass through the pinhole means. Become. As a result, it is possible to irradiate light from the objective optical system toward the sample from the objective optical system without being affected by multimode dispersion in the multimode fiber. Can be generated efficiently.

  Further, the fluorescence generated in the sample is incident on the pinhole means via the objective optical system. Since the pinhole means is made of a material that transmits visible light, the fluorescence passes through the multimode fiber without being blocked by the pinhole means, and is imaged by the imaging means in the optical unit. In this case, since the fluorescence is not blocked by the pinhole means and is propagated by the multimode fiber having a large core diameter, the fluorescence generated in the sample is imaged by the imaging means without waste, and a bright fluorescent image can be obtained. it can. That is, even if the image pickup means is installed in the optical unit connected to the measurement head via the multimode fiber, a clear fluorescent image can be obtained without much deterioration of the image quality, so the measurement head is made sufficiently small. Therefore, it is possible to provide a laser scanning observation apparatus suitable for observing a sample such as an experimental small animal alive.

Further, with this configuration, it is possible to achieve the above action without increasing the number of parts.

The present invention also provides an optical unit including a laser light source that emits ultrashort pulse laser light, an imaging unit that detects return light from the sample, and light that scans the sample with the ultrashort pulse laser light from the laser light source. A measurement head comprising a scanning unit, an objective optical system that forms an image of an ultrashort pulse laser beam scanned by the optical scanning unit on a sample, a multimode fiber that connects the optical unit and the measurement head, and Provided with a measuring head, comprising pinhole means made of a material that transmits visible light and blocks laser light, and the pinhole means is constituted by a pinhole member formed by coating the surface of a glass plate, A second laser light source provided in the optical unit and emitting laser light; and a material provided in the measuring head and blocking the laser light and visible light. That a confocal pinhole member, to provide a laser-scanning examination apparatus comprising an exchange mechanism to selectively exchanged with the confocal pinhole member and the pinhole member.

With such a configuration, only the lowest-order mode ultrashort pulse laser light is extracted from the ultrashort pulse laser light imparted with multimode dispersion that has been propagated by the multimode fiber, and the sample is irradiated. It is possible to simply configure the pinhole means that allows the obtained fluorescence to be guided to the image pickup means with a multimode fiber without blocking. In this case, the pinhole may be relatively large and easy to manufacture.
Further, it is possible to perform confocal observation by operating the exchange mechanism to change from a pinhole member to a confocal pinhole member and making a laser beam emitted from a laser light source incident on the sample. In this case, multi-photon excitation observation using a pinhole member and an ultrashort pulse laser beam and confocal observation can be performed using the same multimode fiber and imaging means, and laser scanning observation The apparatus can be configured simply and compactly.

  Further, a plurality of types of confocal pinholes having different diameters may be provided. In this case, by increasing the diameter of the confocal pinhole, the resolution of the fluorescent image can be reduced, while the amount of detected fluorescence can be increased to obtain a bright fluorescent image.

Furthermore, in the said invention, it is good also as providing the hole member with a larger diameter than the said confocal pinhole member, and the said replacement mechanism selectively exchanging this hole member.
By doing so, the amount of light irradiated to the sample is not limited, and the return light from the sample is not limited. For example, when focusing on the surface of the sample, a bright and clear image is obtained. This is advantageous.

The reference example of the present invention provides a pinhole member made of a material that transmits visible light and blocks laser light.
According to the present invention, only the lowest order mode laser light among a number of transmission mode laser lights is transmitted in one direction, while visible light such as fluorescence incident from multiple directions is allowed to pass through without being blocked. Can do.

  According to the present invention, a sample can be irradiated with an ultrashort pulse laser with a short pulse width emitted from a laser light source to efficiently generate a multiphoton excitation effect, and a loss of fluorescence obtained from the sample can be reduced. As a result, a bright fluorescent image can be obtained, and the measurement head can be configured compactly.

A laser scanning observation apparatus 1 according to an embodiment of the present invention will be described below with reference to FIG.
A laser scanning observation apparatus 1 according to the present embodiment is a multiphoton excitation observation apparatus, and includes a measurement lens provided with an objective lens 2 arranged to face a sample A such as a small experimental animal as shown in FIG. An apparatus main body 6 including a head 3, a laser light source 4 that generates a near-infrared ultrashort pulse laser beam having a pulse width of about 100 fsec (femtoseconds) and a wavelength of about 976 nm, and a fluorescence observation photodetector 5; The multi-mode fiber 7 that connects the apparatus main body 6 and the measuring head 3 is provided.

The measuring head 3 is provided with a condenser lens 8, a collimating lens 9, a laser scanning unit 10, a pupil projection lens 11, and an imaging lens 12.
The condensing lens 8 forms an ultrashort pulse laser beam propagating through the multimode fiber 7 at the first intermediate image B position.
The collimating lens 9 converts the ultrashort pulse laser beam formed with the first intermediate image by the condenser lens 8 into parallel light.

  The laser scanning unit 10 includes, for example, two galvanometer mirrors (not shown) provided so as to be rotatable around two axes orthogonal to each other. The laser scanning unit 10 transmits the ultrashort pulse laser light propagated from the laser light source 4. The deflection direction is changed, and the sample A is scanned in a two-dimensional direction. The pupil projection lens 11 forms an ultrashort pulse laser beam scanned in the two-dimensional direction by the laser scanning unit 10 at the second intermediate image C position. The imaging lens 12 condenses the ultrashort pulse laser beam that forms the second intermediate image C and makes it incident on the objective lens 2.

  The measuring head 3 that accommodates them is attached to a support column 14 that extends upward from the base 13 so as to be movable in the vertical direction, and is fixed to the tip of an arm 16 that can be moved up and down by a fine adjustment mechanism 15. In the figure, reference numeral 17 denotes a stage on which the sample A is placed, and allows rotation around two horizontal directions and a vertical axis.

  In the present embodiment, the filter member 18 is disposed at the position of the first intermediate image B between the condenser lens 8 and the collimating lens 9 of the measuring head 3. For example, as shown in FIG. 2, the filter member 18 includes a pinhole 18 c configured by applying a coating 18 b to one surface of a glass plate 18 a except for a central point. The coating 18b is made of a material that transmits visible light and blocks ultrashort pulse laser light, for example, a dielectric multilayer film. The pinhole 18c is disposed at a position substantially coinciding with the position of the first intermediate image B.

  That is, only the light in the vicinity of the central portion of the ultrashort pulse laser light collected by the condenser lens 8 can pass through the pinhole 18c and reach the laser scanning portion 10 via the collimator lens 9, while The return light returned from the laser scanning unit 10 side passes through the filter member 18 without being blocked, and enters the condenser lens 8.

  The apparatus body 6 includes a collimating lens 19 that converts return light that has returned through the multimode fiber 7 into parallel light, a dichroic mirror 20 that branches fluorescence from the return light converted into parallel light, and the excitation light. And a barrier filter 21 is provided.

The operation of the thus configured laser scanning observation apparatus 1 according to this embodiment will be described below.
According to the laser scanning observation apparatus 1 according to the present embodiment, the ultrashort pulse laser light emitted from the laser light source 4 passes through the dichroic mirror 20 and is incident on the end surface of the multimode fiber 7 by the collimator lens 19. . Then, it propagates through the multimode fiber 7 and enters the measuring head 3, and is condensed by the condenser lens 8, and forms the same intermediate image as the output end of the multimode fiber 7 in the vicinity of the pinhole 18 c of the filter member 18. To do.

  In this case, according to the present embodiment, the filter member is formed with a pinhole in the center, and the periphery of the filter member is blocked from passing the ultrashort pulse laser beam by the coating. Only the central portion is allowed to pass through the filter member 18 and is incident on the laser scanning portion 10.

The ultrashort pulse laser beam that is allowed to pass through the multimode fiber 7 is propagated in various transmission modes. The transmission mode is schematically shown in FIG. FIG. 3 shows the light intensity distribution of the ultrashort pulse laser beam at the emission end of the multimode fiber 7. The hatched portion in the figure is a region where the light intensity is zero. That is, as shown in FIG. 3A, when the ultrashort pulse laser beam in the lowest order transmission mode (ie, LP ₀₁ mode) is propagated, a region where the light intensity distribution becomes zero in the core. On the other hand, in other transmission modes (LP _nm mode, where n ≧ 1, m ≧ 1) (b) and (c), there is a region where the light intensity is zero near the center of the core.

  Therefore, as in the laser scanning observation apparatus 1 according to the present embodiment, the filter member 18 is disposed at the position of the first intermediate image B of the condenser lens 8 that forms an image of the end surface 7a of the multimode fiber 7. As a result, among the ultrashort pulse laser beams of various transmission modes propagated in the multimode fiber 7, only the ultrashort pulse laser beam of the lowest order mode is selectively passed, and the light of other transmission modes is transmitted. Passage can be blocked. Then, the ultrashort pulse laser beam converted into parallel light by the collimator lens 9 and whose deflection direction is changed by the laser scanning unit 10 passes through the pupil projection lens 11, the imaging lens 12, and the objective lens 2 to the sample A. Irradiated.

Thereby, it is possible to prevent the pulse width of the ultrashort pal laser beam from being extended, similarly to the case where it is propagated by the single mode fiber. Therefore, it is possible to irradiate the sample A with an ultrashort pulse laser beam having an original short pulse width emitted from the laser light source 4, and to efficiently generate a multiphoton excitation phenomenon.
When a multiphoton excitation phenomenon is caused at a position where the ultrashort pulse laser beam is focused, fluorescence can be generated at a relatively deep position inside the sample A. The generated fluorescence returns from the surface of the sample A into the objective lens 2, and passes through the imaging lens 12, the pupil projection lens 11, and the laser scanning unit 10 and is passed through the filter member 18.

  In this case, according to the laser scanning observation apparatus 1 according to the present embodiment, the filter member 18 is formed by the coating 18b made of a material that allows fluorescence to pass through, so that the fluorescence is not blocked by the filter member 18. The light enters the multimode fiber 7. Then, after being propagated through the multimode fiber 7, it is detected by the photomultiplier tube 6 provided in the apparatus body 6.

  In other words, the multimode fiber 7 having a large core diameter can propagate the fluorescence generated from the sample A without waste and detect it by the photomultiplier tube 5 outside the measurement head 3, thereby reducing the loss of fluorescence and making it brighter. In addition, a high-resolution fluorescent image can be obtained. In addition, the relatively large photomultiplier tube 5 can be separated from the measurement head 3 while reducing the loss of fluorescence. Therefore, the measuring head 3 can be reduced in size, and the handling of the measuring head 3 when observing the sample A such as an experimental small animal can be facilitated and the operability can be improved.

In the laser scanning observation apparatus 1 according to the present embodiment, the filter member 18 formed by coating the glass plate 18a with the coating 18b is used. Instead, as shown in FIG. 4, the multimode fiber 7 is used. A pinhole 18c may be formed by forming a pinhole 18c at the center by a coating 18b that covers the core 7b while leaving only the center on the end surface 7a.
Compared with the case where the filter member 18 is arranged at the position of the first intermediate image B between the condenser lens 8 and the collimating lens 9 as in the present embodiment, the end face 7a of the multimode fiber 7 is configured. There is a merit that manufacturing is easy because the part can be enlarged.

Next, a laser scanning observation apparatus 30 according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the laser scanning observation apparatus 1 according to the first embodiment described above, and the description will be simplified.

The laser scanning observation apparatus 30 according to the present embodiment is different from the laser scanning observation apparatus 1 according to the first embodiment in that it can switch between multiphoton excitation observation and confocal observation. Yes.
In the present embodiment, the apparatus main body 6 is provided with two laser light sources 4 and 31. The first laser light source 4 is a light source for multiphoton excitation observation that emits an ultrashort pulse laser beam similar to that of the first embodiment. The second laser light source 31 is a light source for confocal observation that generates laser light having a wavelength of 488 nm, for example. A total reflection mirror 32 and a dichroic mirror 33 are arranged at the exit of the second laser light source 31 so that the laser light can be incident on the same optical path as the optical path from the first laser light source 4. Yes.

  Further, the measurement head 3 is provided with a turret 34 between the condenser lens 8 and the collimating lens 9, and the filter member 18 and the confocal pinhole member 35 are selected by rotating the turret 34. It can be used.

  When performing the multi-photon excitation type observation, the filter member 18 of the turret 34 is selected, and the ultrashort pulse laser beam is emitted from the first laser light source 4 to perform the same as in the first embodiment. The fluorescence that propagates back through the multimode fiber 7 is separated from the optical path by the dichroic mirror 20 in the apparatus main body 6, the excitation light is cut by the barrier filter 21, and detected by the photomultiplier tube 5.

  When performing confocal observation, the confocal pinhole member 35 of the turret 34 is selected, and laser light is emitted from the second laser light source 31. The laser light emitted from the second laser light source 31 is incident on the same optical path as the light emitted from the first laser light source 4 via the total reflection mirror 32 and the dichroic mirror 33. Then, it propagates through the multi-mode fiber 7 and enters the measuring head 3, forms an intermediate image B at the position of the confocal pinhole 35 by the condensing lens 8, is scanned by the laser scanning unit 10, and is connected to the pupil projection lens 11. The sample A is irradiated through the image lens 12 and the objective lens 2.

  Since the confocal pinhole 35 is disposed at a position conjugate with the focal position of the objective lens 2, only the fluorescence returning from the sample A is emitted from the focal position of the objective lens 2. Can pass through. Thereby, the confocal fluorescence image inside the sample A in which the focal position of the objective lens 2 is arranged can be obtained. The fluorescence that propagates back through the multimode fiber 7 is separated from the optical path by the dichroic mirror 20 in the apparatus body 6 and detected by the photomultiplier tube 5.

  As described above, according to the laser scanning observation apparatus 30 according to the present embodiment, the first laser light source 4 and the second laser light source 31 are switched, and the filter member 18 and the confocal pinhole are adjusted accordingly. By switching the member 35, it is possible to switch between multiphoton excitation type observation and confocal observation. In this case, the multiphoton excitation observation and the confocal observation can be performed using the same multimode fiber 7 and can be detected using the same photomultiplier tube 5.

  By using the same multimode fiber 7, the number of optical fibers connecting the apparatus main body 6 and the measurement head 3 can be reduced, and the operability of the measurement head 3 can be improved. Moreover, by using the common photomultiplier tube 5, the number of parts can be reduced and the cost can be reduced, and the enlargement of the apparatus main body 6 can be suppressed.

  In the present embodiment, the case where the turret 34 includes the filter member 18 and the single confocal pinhole member 35 has been described as an example. However, the present invention is not limited to this, and the diameter of the pinhole is not limited to this. A plurality of types of confocal pinhole members different from each other may be provided in the turret 34 so as to be selectable. According to the confocal observation using a confocal pinhole member having a larger aperture, although the resolution of the fluorescence image is lowered, the amount of fluorescence passing through the pinhole can be increased accordingly, so that a brighter fluorescence image can be obtained. It can be detected in the photomultiplier tube 5.

  Furthermore, in addition to the confocal pinhole member 18, if the turret 34 is provided with a pinhole member having a very large aperture or a hole member through which all light passes, the objective lens 2 is reflected by the reflected light from the sample A. This is advantageous when focusing.

1 is an overall configuration diagram showing a laser scanning observation apparatus according to a first embodiment of the present invention. It is a longitudinal cross-sectional view which shows the filter member used for the laser scanning observation apparatus of FIG. It is a schematic diagram explaining the transmission mode in a multimode fiber. It is a longitudinal cross-sectional view which shows the modification of the filter member of FIG. It is a whole block diagram which shows the laser scanning type observation apparatus which concerns on the 2nd Embodiment of this invention.

Explanation of symbols

A Sample 1 Laser scanning observation apparatus 2 Objective optical system 3 Measuring head 4 Laser light source 5 Photomultiplier tube (imaging means)
6 Main unit (optical unit)
7 Multimode fiber 10 Laser scanning unit (optical scanning unit)
18 Filter member (pinhole means)
18a glass plate 18b coating 31 second laser light source 34 turret (exchange mechanism)
35 Confocal pinhole members

Claims (4)

An optical unit comprising a laser light source that emits ultrashort pulse laser light and an imaging means for detecting return light from the sample;
A measurement head comprising: an optical scanning unit that scans an ultrashort pulse laser beam from a laser light source onto the sample; and an objective optical system that forms an image of the ultrashort pulse laser beam scanned by the optical scanning unit on the sample;
A multimode fiber connecting these optical units and the measuring head;
Provided in the measurement head, comprising pinhole means made of a material that transmits visible light and blocks laser light ,
The pin hole means, said multimode laser scanning observation apparatus that is formed by coating on an end face of the fiber.   An optical unit comprising a laser light source that emits ultrashort pulse laser light and an imaging means for detecting return light from the sample;
  A measurement head comprising: an optical scanning unit that scans an ultrashort pulse laser beam from a laser light source onto the sample; and an objective optical system that forms an image of the ultrashort pulse laser beam scanned by the optical scanning unit on the sample;
  A multimode fiber connecting these optical units and the measuring head;
  Provided in the measurement head, comprising pinhole means made of a material that transmits visible light and blocks laser light,
The pinhole means is constituted by a pinhole member formed by coating the surface of a glass plate,
  A second laser light source provided in the optical unit and emitting laser light;
  A confocal pinhole member made of a material that is provided in the measurement head and blocks laser light and visible light;
  A laser scanning observation apparatus comprising: an exchange mechanism that selectively exchanges the pinhole member and the confocal pinhole member. The laser scanning observation apparatus according to claim 2 , comprising a plurality of types of confocal pinhole members having different diameters. A hole member having a larger diameter than the confocal pinhole member,
The laser scanning observation apparatus according to claim 2 or 3 , wherein the exchange mechanism selectively exchanges the hole member.

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