JP2014052534A - Confocal microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily and reliably perform detection of fluorescent light by a plurality of light sources with difference optical characteristics without causing cost increase of a device.SOLUTION: By positioning in an optical path B a movable optical element 4 arranged retreatable with respect to the optical path B of first excitation light such as incoherent light, an optical path A of second excitation light such as coherent light is formed, and in a state in which the movable optical element 4 retreats from the optical path B of incoherent light, the optical path A of incoherent light is formed. Accordingly, detection of fluorescent light from a sample 7 by incoherent light and detection of fluorescent light from the sample 7 by coherent light can be performed.

Description

  In the present invention, a disk-shaped micro-aperture rotating disk having a plurality of micro-apertures (a micro-aperture rotating disk indicates a disk with a micro-pattern such as a pinhole or a slit) is used to rotate a sample. More particularly, the present invention relates to a confocal microscope equipped with a plurality of light sources having different light characteristics.

  Conventionally, for example, a pinhole substrate in which minute openings such as a plurality of pinholes are formed, for example, a disk-like shape called a “Nipko Disc” spirally arranged at an interval 10 times the pinhole diameter There is known a confocal microscope that rotates a micro-aperture rotating disk and images light that has passed through a pinhole.

  Confocal microscopes have a confocal effect because pinholes are arranged at conjugate positions with the sample surface, and are characterized by high resolution and high-contrast images compared to other types of optical microscopes. doing.

  In particular, the resolution in the optical axis direction is high, and information on the height direction of the sample surface can be obtained with high accuracy. In addition, scanning with a pinhole on the sample surface is performed by high-speed rotation of a disk having a plurality of pinholes in the microscope field of view. The sample surface can be observed in real time as in

  Recently, multiple stained specimens in which a single specimen is labeled with a plurality of fluorescent dyes have been used. In order to detect fluorescence corresponding to each fluorescent dye for such multiple stained specimens, the detection light from the specimen is dispersed into a plurality of channels for each fluorescent dye, and the detection light (fluorescence) is captured for each channel. Something like that is also considered.

  A confocal scanner microscope shown in Patent Document 1 is known as one that detects fluorescence corresponding to each fluorescent dye for such multiple stained specimens.

  For example, as shown in FIG. 5, the confocal scanner microscope includes a plurality of light sources 11a, 11b, and 11c having different wavelengths, a switching unit 14 that controls passage and blocking of laser light, and a filter 12a having different light transmission bands. , 12b, 12c.

  The laser light that has passed through the switching unit 14 enters the optical fibers 15 a, 15 b, and 15 c, exits from the other end of the optical fiber bundle 15, and enters the mirror 16. When the laser beam reflected by the mirror 16 enters the collimator lens 17, it enters the microlens array disk 18.

  The laser light that has passed through the microlens array disk 18 passes through the pinholes of the dichroic mirror 19c and the minute aperture rotating disk 19, is guided to the sample 19b by the objective lens 19a, and excites the fluorescent dye of the sample 19b. The fluorescence from the sample 19b passes through the objective lens 19a and the pinhole of the minute aperture rotating disk 19 again, is reflected by the dichroic mirror 19c, and is guided to the light detection unit 20.

  The light detection unit 20 includes dichroic mirrors 22a, 22b, and 22c, barrier filters 25a, 25b, and 25c, lenses 24a, 24b, and 24c, and imaging cameras 26a, 26b, and 26c. Fluorescence having a specific wavelength reflected by the dichroic mirrors 22a, 22b, and 22c passes through the barrier filters 25a, 25b, and 25c, is collected by the lenses 24a, 24b, and 24c, and enters the imaging cameras 26a, 26b, and 26c. . Then, the fluorescence is amplified by the imaging cameras 26a, 26b, and 26c, converted into an electrical signal, and output.

JP 2004-212434 A

  By the way, in the confocal scanner microscope of Patent Document 1 described above, it is possible to detect fluorescence corresponding to each fluorescent dye by using a plurality of light sources 11a, 11b, and 11c having different wavelengths.

  However, in such a confocal scanner microscope, it is possible to detect the fluorescence corresponding to each fluorescent dye by the coherent laser light, but the mounted optical components are compatible with the coherent laser light. I am letting. Therefore, there is a problem that a light source such as an LED that emits incoherent light cannot be used.

  Incidentally, as is well known, coherent light and incoherent light have optical characteristics such that their spatiotemporal modulation states are different.

  In order to make an incoherent light source coexist with a coherent light source, it may be possible to arrange a mechanism for switching the optical path of light from each light source. Will increase the cost.

  The present invention has been made in view of such a situation, and a confocal microscope capable of easily and reliably detecting fluorescence with a plurality of light sources having different optical characteristics without increasing the cost of the apparatus. The purpose is to provide.

  A confocal microscope of the present invention is a confocal microscope that detects fluorescence from a sample excited by excitation light that passes through at least a pinhole of a micro-aperture rotating disk, and outputs first excitation light. A light source, a second light source that outputs second excitation light, and a movable optical element that is disposed so as to be movable forward and backward with respect to the optical path of the first excitation light. It is characterized in that the optical path of the second excitation light is formed by being positioned in the optical path of the second excitation light.

  Further, the first excitation light is incoherent light, and the second excitation light is coherent light.

  Further, the movable optical element is characterized in that it is rotatably arranged with one end side as a fulcrum.

  The movable optical element is slidably arranged with one end side as a fulcrum.

  In the confocal microscope of the present invention, the movable optical element disposed so as to be movable back and forth with respect to the optical path of the first excitation light is positioned in the optical path of the first excitation light, so that the optical path of the second excitation light is It is formed. On the other hand, when the movable optical element is retracted from the optical path of the first excitation light, the optical path of the first excitation light is formed.

  Therefore, by switching the position of the movable optical element, it is possible to detect fluorescence from the sample by the first excitation light and detect fluorescence from the sample by the second excitation light.

  Further, for example, when the position of the movable optical element is switched at high speed, even if the first light source and the second light source having different optical characteristics are driven at the same time, the fluorescence from the sample by the first excitation light can be changed. Detection and detection of fluorescence from the sample by the second excitation light can be performed simultaneously.

  Here, the difference in optical characteristics means that, for example, the wavelengths of excitation light are different. Also, for example, the spatial and temporal modulation states are different, such as coherent light such as laser light from a laser light source or incoherent light from a light source such as an LED.

  According to the confocal microscope of the present invention, the detection of fluorescence from the sample by the first excitation light and the detection of fluorescence from the sample by the second excitation light can be performed by switching the position of the movable optical element. Since it was made possible, the fluorescence can be detected easily and reliably by using a plurality of light sources having different light characteristics without increasing the cost of the apparatus.

It is a figure explaining one Embodiment of the confocal microscope of this invention. It is a figure for demonstrating the case where the coherent light from the laser light source of FIG. 1 is output. It is a figure for demonstrating the case where the incoherent light from the LED light source of FIG. 1 is output. It is a figure for demonstrating other embodiment at the time of changing the structure of the movable optical element of FIG. It is a figure for demonstrating an example of the conventional confocal scanner microscope.

  Hereinafter, the details of an embodiment of the confocal microscope of the present invention will be described with reference to FIGS.

  First, as shown in FIG. 1, the confocal microscope includes a laser light source 1 and an LED light source 2. Here, for example, a dichroic mirror 3, a movable optical element 4, a minute aperture rotating disk 5, and an objective lens 6 are arranged on an optical path B indicated by a one-dot chain line from the LED light source 2 to a sample 7 described later.

  An optical path A indicated by a two-dot chain line from the laser light source 1 to the sample 7 described later is positioned on an optical path B indicated by a one-dot chain line from the LED light source 2 to the sample 7 described later as the movable optical element 4 rotates. It is supposed to be formed when.

  In addition, a barrier filter 8 and an imaging camera 9 are arranged on an optical path C indicated by a dotted line of fluorescence described later reflected by the dichroic mirror 3. Note that the fluorescence that will be described later reflected by the dichroic mirror 3 reaches the dichroic mirror 3 from the sample 7 and is reflected by the dichroic mirror 3 as described above, but the fluorescence optical path from the sample 7 to the dichroic mirror 3 Is not shown. The same applies to FIGS. 2 to 4 described below.

  Further, the dichroic mirror 3 is shown as being disposed between the movable optical element 4 and the minute aperture rotating disk 5, but is not limited to this example, and between the LED light source 2 and the movable optical element 4. May be arranged.

  Here, the laser light source 1 outputs coherent laser light (hereinafter referred to as coherent light) that is excitation light. The LED light source 2 outputs incoherent light (hereinafter referred to as incoherent light) that is excitation light. As is well known, coherent light and incoherent light have optical characteristics such that the spatiotemporal modulation states are different.

  Here, although the LED light source 2 is illustrated as what outputs incoherent light, it is not restricted to this, You may use other light sources, such as a halogen lamp and a mercury lamp.

  The dichroic mirror 3 is an optical element for separating coherent light or incoherent light and fluorescence from a sample 7 described later.

  The movable optical element 4 is arranged so as to be movable back and forth with respect to the optical path B of incoherent light, for example. In other words, the movable optical element 4 is rotatable in the direction of the arrow ab with the rotation shaft 4a provided on the lower end side of the movable optical element 4 as a fulcrum. The rotation shaft 4a is rotated by a driving force by the drive mechanism 4b.

  The rotation shaft 4a is not limited to the lower end side of the movable optical element 4, and may be provided on the upper end side. Further, the operation instruction to the drive mechanism 4b may be given by control data from a microcomputer (not shown) or may be given by a switching signal when manually switched by a changeover switch (not shown). However, when the movable optical element 4 is advanced or retracted at high speed with respect to the optical path B, it is preferable to use control data from a microcomputer (not shown).

  When the coherent light from the laser light source 1 is guided to the sample 7 described later, the movable optical element 4 is positioned in the direction of arrow a, that is, on the optical path B of the LED light source 2. Thereby, the optical path A of the laser light source 1 is formed, and the coherent light from the laser light source 1 is reflected to the sample 7 side.

  On the other hand, when incoherent light from the LED light source 2 is guided to the later-described sample 7 side, the movable optical element 4 moves in the direction of the arrow b, that is, the position retracted from the optical path B of the LED light source 2.

  The movable optical element 4 has a property of reflecting incoherent light from the LED light source 2 toward the sample 7 described later and allowing fluorescence from the sample 7 described later to pass through the dichroic mirror 3 as it is. .

  In other words, the movable optical element 4 may be an optical element having both reflection characteristics and transmission characteristics, for example, a beam splitter having both reflection characteristics and transmission characteristics at an arbitrary ratio, and reflection characteristics. And a half mirror with a transmission characteristic of 50:50, either a dichroic mirror that reflects any wavelength or transmits any other wavelength or reflects any other wavelength is used. be able to.

  Further, the positions of the laser light source 1 and the LED light source 2 can be reversed. In this case, when incoherent light from the LED light source 2 is guided to the sample 7 described later, the movable optical element 4 is positioned in the direction of arrow a, that is, on the optical path B described above. On the other hand, when the coherent light from the laser light source 1 is guided to the sample 7 described later, the movable optical element 4 moves in the direction of the arrow b, that is, the position retracted from the optical path B described above.

  The minute aperture rotating disk 5 has a plurality of pinholes (not shown) provided in a spiral shape and rotates at high speed. Thereby, a raster scan on the sample 7 described later is performed by the coherent light or the incoherent light that has passed through the pinhole of the minute aperture rotating disk 5.

  The objective lens 6 focuses the coherent light or incoherent light that has passed through the pinhole of the minute aperture rotating disk 5 on the sample 7. The sample 7 is labeled with a plurality of fluorescent dyes excited by coherent light or incoherent light.

  Then, the fluorescence excited by the coherent light or the incoherent light is reflected to the barrier filter 8 and the imaging camera 9 side by the objective lens 6 and the minute aperture rotating disk 5 dichroic mirror 3.

  Here, the barrier filter 8 is provided in order to improve the S / N ratio of the image, and can remove only wavelengths other than the target wavelength and allow only the target wavelength to enter the imaging camera 9. It is an optical element.

  The imaging camera 9 receives the fluorescence that has passed through the barrier filter 8, converts a captured image thereby into an electrical signal, and outputs the electrical signal. The electrical signal is converted into digital image data and stored in association with the fluorescence color branched by the dichroic mirror 3.

  Next, the operation of the confocal microscope having such a configuration will be described. First, as shown in FIG. 2, when outputting coherent light that is excitation light from the laser light source 1, the movable optical element 4 rotates in the direction of arrow a and is positioned on the optical path B of the LED light source 2. Thereby, the optical path A of the laser light source 1 is formed.

  At this time, the coherent light from the laser light source 1 is reflected by the movable optical element 4 toward the dichroic mirror 3 and the minute aperture rotating disk 5 and passes through the pinhole of the rotating minute aperture rotating disk 5. Is collected on the sample 7. Thereby, a raster scan on the sample 7 is performed.

  At this time, the fluorescence from the fluorescent dye excited on the sample 7 is reflected to the barrier filter 8 and the imaging camera 9 side by the objective lens 6 and the minute aperture rotating disk 5 dichroic mirror 3.

  From the sample 7, the reflected light due to the reflection of the coherent light also passes through the objective lens 6 and the minute aperture rotating disk 5 and reaches the dichroic mirror 3. However, due to the characteristics of the dichroic mirror 3, the reflected light is not reflected to the barrier filter 8 and the imaging camera 9 side.

  Here, when the fluorescence that has passed through the barrier filter 8 is captured by the imaging camera 9, the captured image is converted into an electrical signal and output. Then, the electrical signal is converted into digital image data and stored in association with the fluorescence color branched by the dichroic mirror 3.

  On the other hand, as shown in FIG. 3, when outputting incoherent light that is excitation light from the LED light source 2, the movable optical element 4 rotates in the direction of the arrow b and retracts from the optical path B of the LED light source 2. In this case, the optical path A of the laser light source 1 described above is eliminated.

  At this time, the incoherent light from the LED light source 2 passes through the dichroic mirror 3 and further passes through the pinhole of the rotating micro-aperture rotating disk 5 and is collected on the sample 7 by the objective lens 6. Thereby, the raster scan on the sample 7 is performed as described above.

  At this time, the fluorescence from the fluorescent dye excited on the sample 7 passes through the objective lens 6 and the minute aperture rotating disk 5 and is reflected by the dichroic mirror 3 toward the barrier filter 8 and the imaging camera 9 as described above. .

  From the sample 7, similarly to the above, the reflected light due to the reflection of the incoherent light passes through the objective lens 6 and the minute aperture rotating disk 5 and reaches the dichroic mirror 3, but due to the characteristics of the dichroic mirror 3. The light is not reflected toward the barrier filter 8 and the imaging camera 9 side.

  Here, as described above, when the fluorescence that has passed through the barrier filter 8 is captured by the imaging camera 9, the captured image is converted into an electrical signal and output. Then, the electrical signal is converted into digital image data and stored in association with the fluorescence color branched by the dichroic mirror 3.

  In addition, although demonstrated here as the case where the output of the coherent light from the laser light source 1 and the output of the incoherent light from the LED light source 2 are performed separately, coherent light and It is also possible to detect each fluorescence corresponding to incoherent light.

  In this case, as shown in FIG. 1, the rotation of the movable optical element 4 to the arrow ab is switched at high speed. Thereby, for example, if the fluorescence is detected in synchronization with the switching timing of the movable optical element 4, it is possible to detect the respective fluorescence corresponding to the coherent light and the incoherent light.

  As described above, in the present embodiment, the movable optical element 4 that is disposed so as to be able to advance and retreat with respect to the optical path B of the first excitation light, for example, incoherent light, is positioned in the optical path B. For example, in the state where an optical path A of coherent light, which is light, is formed and the movable optical element 4 is retracted from the optical path B of incoherent light, the optical path A of incoherent light is formed.

  Thus, by switching the position of the movable optical element 4, it is possible to detect fluorescence from the sample 7 using incoherent light and to detect fluorescence from the sample 7 using coherent light.

  For example, when the position of the movable optical element 4 is switched at high speed, the first light source, for example, the LED light source 2 and the second light source, for example, the laser light source 1 can be driven simultaneously. Detection of fluorescence from the sample 7 by coherent light and detection of fluorescence from the sample 7 by coherent light can be performed simultaneously.

  Here, the difference in optical characteristics means that, for example, the wavelengths of excitation light are different. Also, for example, the spatial and temporal modulation states are different, such as coherent light such as laser light from a laser light source or incoherent light from a light source such as an LED.

  In the present embodiment, the movable optical element 4 is rotated in the direction of the arrow ab so that the movable optical element 4 can move forward and backward with respect to the optical path B of the incoherent light, for example. Not limited to this, the advance / retreat operation of the movable optical element 4 can be changed.

  In this case, for example, as shown in FIG. 4, a movable shaft 4d is provided on the lower end side of the movable optical element 4, and the movable shaft 4d is slidably arranged in the direction of arrows ab. Then, the movable optical element 4 is slid to the arrows ab with the movable shaft 4d as a fulcrum by the driving force of the drive mechanism 4c. Thereby, the movable optical element 4 can be made to advance and retreat with respect to the optical path B of incoherent light, for example.

  In the present embodiment, the laser light source 1 and the LED light source 2 are described as one, but the present invention is not limited to this example, and a plurality of laser light sources 1 and LED light sources 2 may be provided. In this case, the laser light source 1 and the LED light source 2 may be switched so as to be positioned in the optical paths A and B described above.

  In the present embodiment, the microlens array disk 18 shown in FIG. 5 can be arranged close to the minute aperture rotating disk 5. In this case, the movable optical element 4 described above may be disposed between the minute aperture rotating disk 5 and the microlens array disk 18 shown in FIG.

DESCRIPTION OF SYMBOLS 1 Laser light source 2 LED light source 3 Dichroic mirror 4 Movable optical element 4a Rotating shaft 4b, 4c Drive mechanism 4d Movable shaft 5 Micro aperture turntable 6 Objective lens 7 Sample 8 Barrier filter 9 Imaging camera A-C Optical path

Claims (4)

  A confocal microscope for detecting fluorescence from a sample excited by excitation light passing through at least a minute aperture of a minute aperture rotating disk, the first light source outputting the first excitation light, and the second A second light source that outputs excitation light; and a movable optical element that is disposed so as to be movable back and forth with respect to the optical path of the first excitation light. The movable optical element is positioned in the optical path of the first excitation light. By doing so, an optical path of the second excitation light is formed.   The confocal microscope according to claim 1, wherein the first excitation light is incoherent light, and the second excitation light is coherent light.   The confocal microscope according to claim 1, wherein the movable optical element is rotatably arranged with one end side as a fulcrum.   The confocal microscope according to claim 1, wherein the movable optical element is slidably arranged with one end side as a fulcrum.

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