JP2005085885A - Laser light source apparatus and confocal microscope apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a laser light source apparatus which can easily exchange a laser light source apparatus, and to provide a confocal microscope apparatus utilizing the same laser light source apparatus. <P>SOLUTION: The laser light source apparatus 1 is provided with laser light sources 11, 21 and 31 which respectively oscillates laser beams L10, L20 and L30 of different wavelengths; a composite optical system 2 for selecting the light of the predetermined wavelength from among the laser beams L10, L20 and L30, and then compositing a plurality of laser beams to only one laser beam L1; an optical fiber 5 for introducing the laser beam L1, and then propagating this light beam L1 to the external side; and base members 10, 20 and 30 for holding by each pair of the laser light sources 11, 21 and 31 and dichroic mirrors 14, 24 and 34 for selecting the light beam of the predetermined wavelength, capable of mutually loading and unloading with a coupling portion. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a laser light source device used in a confocal microscope apparatus.

  In recent years, among optical microscopes, a scanning confocal microscope not only having a high lateral resolution but also a high vertical resolution has attracted attention. In a scanning confocal microscope, laser light from a laser light source is guided to an illumination optical system and forms an image on a specimen as excitation light through an objective lens. The spot light on the specimen is observed as an observation image by being scanned two-dimensionally. When a sample is irradiated with laser light, reflection, absorption, fluorescence, scattering, and the like occur due to the optical characteristics of the sample.

  Recently, multiple excitation observation has been performed in which a biological specimen stained with a plurality of fluorescent reagents is simultaneously irradiated with a plurality of wavelengths of laser light for observation. In the prior art, the light source unit mounts a plurality of laser light sources with different wavelengths on the base unit, selects the wavelength of each laser beam using a dichroic mirror, aligns the optical axes to one, and then attaches to the optical fiber. Incident. (For example, refer to Patent Document 1).

JP 2002-221663 A (page 3, FIG. 2)

  In order to compare and observe various tissues of a biological specimen, an appropriate wavelength of laser light must be selected according to the purpose of observation. However, the space of the light source unit in the microscope apparatus is limited. Conventionally, for example, three types of typical laser light sources are selected from various laser light sources having various oscillation wavelengths and used by being attached to the base portion.

  As described above, since only a laser light source having a representative wavelength has been installed in the past, the sample may not be irradiated with light having the highest excitation efficiency. There was a problem that could not be obtained. The present invention provides a laser light source device that can irradiate a laser beam in accordance with a fluorescent reagent to be used, and a confocal microscope device using the laser light source device.

  (1) The laser light source unit of claim 1 holds a pair of a laser light source, a wavelength selection optical element that selects light of a predetermined wavelength from laser light oscillated by the laser light source, and a laser light source and a wavelength selection optical element. And a base member that is provided on the base member and holds a pair of other laser light sources and wavelength selection optical elements, and is detachably connected to the base member.

(2) In the laser light source device according to claim 2, a plurality of laser light sources that respectively oscillate a plurality of laser beams having different wavelengths, and light of a predetermined wavelength emitted from the plurality of laser light sources are wavelength selection optical elements. And a combining optical system that combines a plurality of laser beams having the selected wavelength into one, an optical fiber that introduces the combined laser beam into one and propagates to the outside, a laser light source, and The wavelength selection optical element is held for each pair, and includes a plurality of base members that can be attached to and detached from each other by a connecting portion.
The plurality of base members of the above laser light source device are arranged such that the base member holding the laser light source having the shortest oscillation wavelength is arranged closest to the laser light entrance with the laser light entrance of the optical fiber as a reference, and the oscillation wavelength is short. It is preferable to connect in order.
The plurality of wavelength selection optical elements are preferably dichroic mirrors that reflect a plurality of laser beams each having a selected wavelength and transmit a laser beam having a wavelength longer than the vicinity of the selected wavelength.

  (3) A confocal microscope apparatus according to claim 5 is capable of observing multiple excitation fluorescence with respect to the laser light source device, an illumination optical system for introducing laser light through an optical fiber of the laser light source device, and a specimen. And a detection optical system.

  According to the present invention, a laser light source and an optical element suitable for the laser light source are mounted on the base member one by one. Therefore, a laser light source having an oscillation wavelength suitable for the fluorescent reagent to be used can be easily replaced, and high intensity fluorescence Can be obtained.

Hereinafter, a laser light source apparatus and a confocal microscope apparatus according to the present invention will be described with reference to FIGS. 1-4, the same code | symbol is attached | subjected to the same component.
FIG. 1 is a configuration diagram showing the overall configuration of a confocal microscope apparatus according to an embodiment of the present invention. The confocal microscope apparatus according to the present embodiment includes a laser light source device 1 and a scanning confocal microscope 100. FIG. 1 schematically shows the laser light source device 1 in an enlarged state.

-Laser light source device-
The laser light source device 1 includes laser light sources 11, 21, 31 (in order of shorter oscillation wavelengths), a combining optical system 2, a base plate 3, and a single mode optical fiber 5. The three laser light sources 11, 21, 31 are mounted on three base plates 10, 20, 30, respectively. The combining optical system 2 includes shutters 12, 22, 32, ND filters 13, 23, 33, dichroic mirrors 14, 24, 34, and a fiber coupler 4. The shutter 12, the ND filter 13, and the dichroic mirror 14 are placed on the base plate 10. Similarly, the shutter 22, the ND filter 23, and the dichroic mirror 24 are placed on the base plate 20, and the shutter 32, the ND filter 33, and the dichroic mirror 34 are placed on the base plate 30. The fiber coupler 4 is placed on the base plate 3 and connected to the single mode optical fiber 5.

The structure of the base plates 10 and 20 shown in FIG. 1 will be described with reference to FIG.
FIG. 2 is a schematic diagram showing the structure of the base portion on which the individual laser light sources shown in FIG. 1 are placed. 2A is a top view of the base plates 10 and 20, and FIG. 2B is a side view thereof. Two pins 10a are planted on the side surface of the base plate 10 and two holes 10b are formed on the opposite side surfaces. Two pins 20a are also planted on the side surface of the base plate 20 and face each other. Two holes 20b are formed on the side surface. The interval between the two pins 10a and the interval between the two holes 10b are the same. In addition, through holes (not shown) are formed in the base plates 10 and 20.

  The holding portion 16 is provided on the two rails 16a extending in the direction perpendicular to the paper surface in FIG. 2B, a support member 16b for supporting the two rails 16a in parallel, and the lower surfaces of the two rails 16a. The elastic member 16c. The bolt 15 is used to fix each base plate to the rail 16a.

  When connecting the base plates 10 and 20, the base plates 10 and 20 are placed on the rail 16a, the pins 20a of the base plate 20 are fitted into the holes 10b of the base plate 10, and the bolts 15 are inserted into the through holes. Through, the base plates 10 and 20 are fixed to the rail 16a. On the contrary, when separating the base plates 10 and 20, the bolt 15 is removed and the pin 20a is pulled out from the hole 10b. Thus, the connecting operation and the separating operation are easy.

  Since each base plate has the same interval between two pins and the interval between the two holes, all the base plates can be connected to each other. Further, since the connection between the pin and the hole portion also serves as positioning, optical adjustment after the connection operation is not necessary. Although not shown, the base plate 3 on which the fiber coupler 4 is placed also has two holes formed at the same interval, and two pins on each base plate on which the laser light source is placed. It can be made to fit.

  Referring to FIG. 1 again, the laser light L10 emitted from the laser light source 11 enters the dichroic mirror 14 through the shutter 12 that opens the optical path and the ND filter 13 that adjusts the light amount. The dichroic mirror 14 selects light in the vicinity of the center wavelength having a high oscillation intensity among the oscillation wavelengths of the laser light L10, reflects the light rightward on the drawing, and guides it to the fiber coupler 4. At this time, the dichroic mirror 14 transmits light on the longer wavelength side than light near the center wavelength upward in the drawing.

  Similarly, the laser light L20 emitted from the laser light source 21 enters the dichroic mirror 24 through the shutter 22 and the ND filter 23. The light in the vicinity of the selected wavelength of the laser light L20 is reflected to the right by the dichroic mirror 24, passes through the dichroic mirror 14 and enters the fiber coupler 4, and is longer than the vicinity of the selected wavelength of the laser light L20. The light is transmitted upward.

  The laser light L30 emitted from the laser light source 31 enters the dichroic mirror 34 through the shutter 32 and the ND filter 33. Light in the vicinity of the selected wavelength of the laser light L30 is reflected to the right by the dichroic mirror 34, sequentially passes through the dichroic mirrors 24 and 14, and enters the fiber coupler 4. Light on the longer wavelength side than the vicinity of the selected wavelength of the laser light L30 is transmitted upward. However, since the dichroic mirror 34 is located on the longest wavelength side (the left end in the figure) and light transmission is not essential, it may be replaced with a total reflection mirror.

  The laser beams L10, L20, and L30 thus selected as wavelengths become one beam, enter the fiber coupler 4 as the laser beam L1, and after being condensed by the condenser lens 4a, the single mode optical fiber 5 is collected. Is incident on. The wavelength-selected laser beams L10, L20, and L30 can be adjusted to be on the same axis by changing the positions and angles of the dichroic mirrors 14, 24, and 34, respectively.

  FIG. 3 is a schematic view illustrating various laser light sources placed on the base plate. As illustrated, each laser light source, each dichroic mirror, and the like are mounted on each base plate, and each laser light source has various sizes and shapes depending on the oscillation wavelength. Each dichroic mirror also has unique spectral characteristics.

  The laser light source 51 is a violet-LD that outputs a laser beam L50 having a center wavelength of 405 nm. Similarly, the laser light source 61 is a DPSS laser (L60: 430 nm), the laser light source 11 is an air-cooled Ar laser laser (L10: 488 nm and 514 nm), the laser light source 21 is a G / He-Ne laser (L20: 543 nm), and a laser light source 31. Is a Y / He-Ne laser (L30: 594 nm), and the laser light source 41 is an R / He-Ne laser (L40: 633 nm).

-Scanning confocal microscope-
As shown in FIG. 1, the scanning confocal microscope 100 has a configuration of an epi-fluorescence microscope, and includes an illumination optical system 110 and a detection optical system 120. The illumination optical system 110 is connected to the laser light source device 1 by a single mode optical fiber 5. The illumination optical system 110 is disposed in the order of the beam expander 111, the beam splitter 112, the optical scanning unit 113, the scanning lens 114, the relay lens 116, and the objective lens 118 along the optical path of the laser light L1. The optical scanning unit 113 is provided with a pair of movable total reflection mirrors 113a and 113b which will be described later with reference to FIG.

  The detection optical system 120 includes an objective lens 118, a relay lens 116, a scanning lens 114, a light scanning unit 113, a beam splitter 112, a light detection lens 119, a light blocking plate 121 with a pinhole, The mode optical fiber 122 and the photodetector 130 are arranged in this order. The photodetector 130 includes a collimator lens 131, a dichroic mirror 132, barrier filters 133 and 134, and photodetector elements 135 and 136. The photodetector 130 is electrically connected to the image processing unit 140.

Hereinafter, the illumination optical system 110 and the detection optical system 120 will be described in detail with reference to FIG.
FIG. 4 is a diagram for explaining the optical system of the scanning confocal microscope according to the embodiment of the present invention. The laser light L1 from the laser light source device 1 enters the beam expander 111 through the single mode optical fiber 5. As described above, the laser light L1 is a single beam including light of a plurality of wavelengths. The laser beam L1 is enlarged to a desired beam diameter by the beam expander 111 and enters the optical scanning unit 113. The laser beam L1 is two-dimensionally scanned in two directions orthogonal to the optical axis by changing the tilt angle in conjunction with the total reflection mirrors 113a and 113b, and formed on the primary image plane 115 by the scanning lens 114. The image is formed as a point image on the biological specimen S through the relay lens 116, the mirror 117, and the objective lens 118. Since this point image is focused to a size determined by the NA of the objective lens 118, phenomena such as reflection, absorption, fluorescence generation, and scattering are caused in the irradiated region due to the optical characteristics of the biological specimen S at this point. Arise. The mirror 117 is not essential because it simply changes the direction of the laser beam L1.

  Fluorescence or reflected light from the biological specimen S passes through the objective lens 118 and the mirror 117, forms an image on the primary image plane 115 by the relay lens 116, then becomes parallel light by the scanning lens 114 and enters the optical scanning unit 113. The fluorescence from the biological specimen S is always returned to the same optical path as the laser beam L1 incident as illumination light by being scanned by the optical scanning unit 113, that is, descanned.

  The fluorescence that has entered the beam splitter 112 from the optical scanning unit 113 is reflected by the beam splitter 112 and collected on the light shielding plate 121 by the light detection lens 119. A pinhole 121a is formed in the light shielding plate 121. Since the light shielding plate 121 and the biological specimen S are in a conjugate positional relationship, an image of the fluorescence emission point on the biological specimen S is formed at the position of the pinhole 121a. Only the light that has passed through the pinhole 121 a reaches the photodetector 130 via the multimode optical fiber 122. Even if there is light generated from other points on the biological specimen S, it is blocked by the light shielding plate 121 and does not reach the photodetector 130. As a result, in the scanning confocal microscope, it is possible to observe the specimen not only with high lateral resolution but also with high vertical resolution.

  The fluorescence that has reached the photodetector 130 enters the dichroic mirror 132 through the collimator lens 131, is separated into two types of wavelengths of fluorescence, is incident on the barrier filters 133 and 134, and is detected by the photodetectors 135 and 136. Detected. In FIG. 4, two pairs of barrier filters and light detection elements are shown, but three or more sets can be provided depending on the type of wavelength to be detected.

  Hereinafter, a method of using the confocal microscope apparatus of the present embodiment, particularly a laser light source replacement operation, will be described with reference to FIGS. 5 and 6, the same components as those in FIGS. 1 and 3 are denoted by the same reference numerals, and the description thereof is omitted.

  FIG. 5 is a configuration diagram of a laser light source device in which base plates on which the six types of laser light sources shown in FIG. 3 are respectively placed are arranged from the right to the left in order of the oscillation wavelength of the laser light source. In this case, since multiple types of laser light sources are equipped, multiple excitation observations corresponding to the types of fluorescent reagents for staining the biological specimen S can be performed at most. On the other hand, since the laser light source that does not participate in the excitation of the selected fluorescent reagent is still provided, the laser light source device becomes large. Therefore, if only the laser light source necessary for fluorescence generation is provided, the laser light source device can be made compact.

FIG. 6 is a configuration diagram of a laser light source device in which base plates on which three types of laser light sources are respectively placed are arranged from right to left in order of increasing oscillation wavelength of the laser light source.
As shown in FIG. 6A, the laser light source device 1 includes an air-cooled Ar laser 11 (488 nm and 514 nm), a G / He-Ne laser 21 (543 nm), and a Y / He-Ne laser 31 (594 nm). Already installed. As a procedure for attaching the laser light source unit, the base plate 10 may be attached to the base plate 3, the base plate 20 may be attached to the base plate 10, and the base plate 30 may be attached to the base plate 20 by fitting pins and holes.

  Now, when Acridine Orange is selected as the fluorescent reagent for staining the biological specimen S, an excitation / fluorescence spectrum diagram as shown in FIG. 7 is obtained. For the excitation spectrum curve, the absorption on the left vertical axis is used, and for the fluorescence spectrum curve, the fluorescence intensity on the right vertical axis is used. The larger the absorption amount of the excitation spectrum curve, the higher the excitation efficiency.

  When Acridine Orange is used as a fluorescent reagent, the excitation efficiency is maximized near the wavelength of 430 nm. However, in the combination of the air-cooled Ar laser 11, the G / He-Ne laser 21, and the Y / He-Ne laser 31 (FIG. 6A), the oscillation wavelength of the air-cooled Ar laser 11 having the shortest wavelength is 488 nm. Excitation efficiency is very low. When the excitation efficiency is low, the output of the laser beam must be increased in order to generate brighter fluorescence, but irradiating the biological specimen S with high-intensity laser light damages the biological specimen S (fading). Will interfere with accurate observation.

Therefore, it is desirable to use the DPSS laser 61 (430 nm) from the viewpoint of excitation efficiency. For example, as shown in FIG. 6B, when the combination of the DPSS laser 61, the air-cooled Ar laser 11, and the G / He-Ne laser 21 is selected, the laser light source needs to be replaced. The arrangement after the replacement is the DPSS laser 61, the air-cooled Ar laser 11, and the G / He-Ne laser 21 in the order from the fiber coupler 4.
In this way, since only a laser light source having a required wavelength can be set at the time of observation according to the excitation wavelength of the fluorescent reagent to be used, the space occupied by the laser light source device can be reduced.

As a procedure for exchanging the laser light source unit as shown in FIG. 6A to FIG. 6B, (1) the base plate 30 is removed, (2) the base plates 10 and 20 are shifted to the left side, and (3) the base A base plate 60 is mounted in the space between the plates 10 and 3. Thus, the replacement work of the laser light source is easy.
Since the base plates are connected to each other by fitting the above-described pins and holes, the three laser beams L60, L10, and L20 become one laser beam L1 with the optical axis aligned, and pass through the fiber coupler 4. The light enters the single mode optical fiber 5. Therefore, positioning and optical axis adjustment are not necessary. Of course, another method may be used as long as the optical axis does not shift without using the fitting between the pin and the hole to connect the base plates. For example, a positioning member for positioning each base plate may be provided on the rail, and the positional relationship between the base plates may be determined.

  In the present embodiment, the laser light source device in which the laser light source having the short oscillation wavelength is arranged near the fiber coupler 4 and arranged in the order from the shortest oscillation wavelength has been described. However, the laser light source device may not necessarily be arranged in the order from the shortest oscillation wavelength. For example, when there are three laser light source units having wavelengths λ1, λ2, and λ3 (where λ1 <λ2 <λ3) are selected and arranged in the order of λ2, λ1, and λ3 from the vicinity of the fiber coupler 4, the dichroic mirror The spectral characteristics may be set as follows. That is, the dichroic mirror of the laser light source unit having the selected wavelength λ2 is set to reflect light in the vicinity of the selected wavelength λ2 and transmit both light having a longer wavelength and light having a shorter wavelength than the vicinity of the selected wavelength λ2.

  Further, in the present embodiment, the scanning confocal microscope 100 is configured as an epi-illumination microscope, and fluorescence observation has been described. However, the present embodiment can also be applied to observation of reflected images and detection of scattered light. Furthermore, if a transmission microscope is used instead of the epi-illumination microscope, it can be applied to transmission image observation that takes into account absorption of transmitted light in addition to fluorescence observation and scattered light detection. Reflection, transmission, absorption, fluorescence, scattering, and the like by a biological specimen have properties that depend on the wavelength of irradiation light.

It is a block diagram which shows the whole structure of the confocal microscope which concerns on embodiment of this invention. It is a figure which shows the structure of the base board which mounts each laser light source shown in FIG. It is a schematic diagram which illustrates the various laser light sources mounted in the base board. It is a figure explaining the optical system of the scanning confocal microscope which concerns on embodiment of this invention. It is a block diagram of a laser light source device when six types of laser light sources are arranged. It is a block diagram of the laser light source device when three types of laser light sources are arranged. FIG. 6 is an excitation / fluorescence spectrum diagram for the fluorescent reagent Acridine Orange.

Explanation of symbols

1: Laser light source device 2: Synthetic optical system 3, 10, 20, 30, 40, 50, 60: Base plate 4: Fiber coupler 5: Single mode fiber 10, 20, 30, 40, 50, 60: Laser light source 14 , 24, 34, 44, 54, 64: Dichroic mirror 100: Scanning confocal microscope 110: Illumination optical system 120: Detection optical system S: Biological specimen L1, L10, L20, L30, L40, L50, L60: Laser light

Claims (5)

A laser light source;
A wavelength selection optical element that selects light of a predetermined wavelength from laser light oscillated by the laser light source;
A base member holding a pair of the laser light source and the wavelength selecting optical element;
A laser light source unit, comprising: a base member that is provided on the base member and holds a pair of another laser light source and a wavelength selection optical element; A plurality of laser light sources that respectively oscillate a plurality of laser beams having different wavelengths;
A combining optical system that selects light of a predetermined wavelength emitted from the plurality of laser light sources by each wavelength selection optical element, and combines a plurality of laser beams having the selected wavelength into one;
An optical fiber that introduces the laser beam combined into the one and propagates to the outside;
A laser light source device comprising: a plurality of base members that hold the laser light source and the wavelength selection optical element for each pair and are detachable from each other by a connecting portion. The laser light source device according to claim 2,
The plurality of base members are arranged in such a manner that a base member holding a laser light source having the shortest oscillation wavelength is disposed closest to the laser light entrance with reference to the laser light entrance of the optical fiber, and is connected in order of shortest oscillation wavelength. A laser light source device. In the laser light source device according to claim 2 or 3,
The plurality of wavelength selection optical elements are dichroic mirrors that reflect a plurality of laser beams each having the selected wavelength and transmit a laser beam having a wavelength longer than the vicinity of the selected wavelength. Laser light source device. A laser light source device according to any one of claims 2 to 4,
An illumination optical system that introduces the laser beam synthesized into one through the optical fiber of the laser light source device and irradiates the specimen with the laser beam as excitation light; and
A confocal microscope apparatus comprising a detection optical system capable of observing multiple excitation fluorescence with respect to the specimen.

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