JP4593141B2 - Optical scanning observation device - Google Patents

Description

  The present invention relates to an optical scanning observation apparatus.

Conventionally, as this type of optical scanning observation apparatus, the one shown in Patent Document 1 is known.
This optical scanning observation apparatus includes a scanning laser microscope composed of a fluorescence microscope, an optical fiber bundle in which one end surface is disposed on a focal plane of a first objective lens included in the scanning laser microscope, and the optical fiber bundle. And a second objective lens arranged so that the other end surface is a light source.

According to this optical scanning observation apparatus, the end face of the optical fiber bundle is made to function as a confocal pinhole, and the fluorescent light is generated at the focal position by the laser beam condensed at the focal position of the second objective lens, Internal organization can be acquired.
Further, this Patent Document 1 also discloses a confocal scanner in which a condensing disk and a pinhole disk are connected and rotated by a motor. By employing this confocal scanner, it is possible to scan a plurality of spots on the sample at a time and obtain a confocal image at high speed.
JP 2003-344777 A (FIG. 1 etc.)

  However, since the optical scanning observation apparatus disclosed in Patent Document 1 employs a single pinhole disk including a fixed confocal pinhole or a pinhole having a predetermined pattern, an observable region However, it is limited to a very thin and limited depth range below the surface of the living body. For this reason, there is an inconvenience that it is difficult to detect and focus an observation target region. In order to avoid this, it is conceivable to prepare and replace a plurality of confocal pinholes and pinhole disks, but there is a disadvantage that the operability is poor and troublesome. In addition, depending on the observation target, it may be preferable to obtain a fluorescent image having a deep depth and a bright image even when the resolution is lowered.

  The present invention has been made in view of the circumstances described above, and can freely change the resolution of an image obtained with a simple configuration, and can change the brightness and observation depth of a fluorescent image in accordance with the purpose of observation. An object of the present invention is to provide an optical scanning confocal observation device.

In order to achieve the above object, the present invention provides the following means.
A light source unit, a condensing lens that forms a first intermediate image of the excitation light emitted from the light source unit in a line, an imaging lens that condenses the intermediate image, and the imaging lens. A first objective lens that forms a second intermediate image of the excitation light, an optical fiber bundle having one end face in the vicinity of the second intermediate image position, and the other end face side of the optical fiber bundle. A second objective lens that forms an image of light emitted from the other end face of the fiber bundle on the sample, and an imaging that picks up the return light returning through the second objective lens, the optical fiber bundle, the first objective lens, and the imaging lens and means, in the first intermediate image position, selectively turned on with receiving the first intermediate image at the same time, the scanning mirror device formed by at least one row arranging a plurality of mirrors that can be turned off is arranged, the scanning The light reflected by each mirror constituting the mirror element Providing an optical scanning observation apparatus provided with a Galvano mirror for scanning in a direction crossing the row direction of the mirror.

  According to the present invention, the excitation light emitted from the light source unit is condensed by the condenser lens to form a first intermediate image. Since the scanning mirror element is disposed at the position of the first intermediate image, the excitation light that forms the first intermediate image is partially turned on and off by selectively turning on and off the plurality of mirrors constituting the scanning mirror element. And the sample can be irradiated through the imaging lens, the first objective lens, the optical fiber bundle, and the second objective lens. Return light such as fluorescence generated in the sample returns through the second objective lens, the optical fiber bundle, the first objective lens, and the imaging lens, and only the reflected light from the mirror in the ON state of the scanning mirror element is image pickup means. To be imaged.

  Then, by sequentially switching the mirrors that are turned on among the mirrors constituting the scanning mirror element, the irradiation position of the excitation light to the sample can be changed, and a wide range can be observed. By making each mirror that constitutes the scanning mirror element sufficiently small, each mirror can function as a confocal pinhole, and the confocal point of the observation target that extends to a predetermined depth below the surface of the sample. A fluorescent image can be obtained.

In this case, since the on / off state of the mirror of the scanning mirror element can be arbitrarily changed, by switching the on / off state, the same effect as that of exchanging a plurality of confocal pinhole members can be achieved. In other words, if you want to obtain a high-resolution confocal image, reduce the number of mirrors that are turned on at a time, and if you want to obtain a bright and deep image, turn on multiple adjacent mirrors simultaneously. It can respond by doing.
Further, since the excitation light is scanned in one direction by the operation of the scanning mirror element and the excitation light is scanned in the direction intersecting with the galvano mirror, the excitation light can be scanned two-dimensionally on the sample. In this case, by forming the first intermediate image in a line shape, the excitation light emitted from the light source unit can be concentrated in a narrow region as compared with the case of forming the two-dimensional first intermediate image. So a bright image can be obtained.

In the said invention, it is preferable to provide the control apparatus which controls the operation pattern of each mirror which comprises a scanning mirror element.
By controlling each mirror with a predetermined operation pattern by the operation of the control device, it is possible to select a desired observation form and perform observation according to the observation object. Further, the operation pattern can be changed easily and at high speed by the control device.

  Moreover, in the said invention, it is preferable to arrange | position so that each mirror which comprises the said scanning mirror element, and the end surface of each fiber core which comprises the said optical fiber bundle may correspond to 1: 1. By doing so, the resolution is not limited by either the scanning mirror element or the optical fiber bundle, and light can be efficiently propagated.

Further, in the above invention, the mirrors constituting the scanning mirror element and the end faces of the fiber cores constituting the optical fiber bundle may be arranged so as to correspond one-to-many or many-to-one. Good.
By doing so, the resolution is limited by either the scanning mirror element or the optical fiber bundle, but the confocal pinhole effect in the scanning mirror element is reduced, and the image is bright and deep. Can be obtained.

  According to the present invention, it is possible to freely change the resolution of an image to be obtained with a simple configuration, and it is possible to change the brightness and observation depth of a fluorescent image according to the purpose of observation.

An optical scanning observation apparatus according to an embodiment of the present invention will be described below with reference to FIGS. 1 and 2.
As shown in FIG. 1, the optical scanning observation apparatus 1 according to the present embodiment has an apparatus main body 3 including a first objective lens 2 and an end face 4 a disposed at an imaging position of the first objective lens 2. An optical fiber bundle 4 and a second objective lens 5 that is disposed on the other end face 4b side of the optical fiber bundle 4 and forms an image of light emitted from the other end face 4b on the sample A are provided.

  The apparatus main body 3 is emitted from a light source unit 6 that generates excitation light, an imaging unit 8 that includes an imaging unit 7 such as a photomultiplier tube that images return light returned from the sample A, and a light source unit 6. And an optical scanning unit 9 for two-dimensionally scanning the excitation light.

  The light source unit 6 includes a white light source 10 such as a xenon lamp, an excitation filter 11 that transmits white light emitted from the white light source 10 to generate excitation light having an excitation wavelength, and is emitted from the excitation filter 11. A condensing lens 12 for condensing the excitation light and a field stop 13 for limiting the illumination range. Further, the light source unit 6 is provided with a condensing lens 14 that condenses the excitation light transmitted through the field stop 13 and forms a first intermediate image.

  The imaging unit 8 captures the return light, a barrier filter 15 that transmits reflected light and fluorescence returning from the sample A side and blocks excitation light, a photometric filter 16 that measures the amount of return light, and the like. And a focusing lens 17 that forms an image on the means 7. The imaging unit 8 is provided with a condenser lens 18 that condenses the return light emitted from the first intermediate image position B and converts it into parallel light.

  The incident optical axis of the excitation light from the light source unit 6 and the optical axis of the image sensor 7 are arranged so as to intersect each other, and the dichroic that selectively reflects the excitation light and transmits other return light at the intersection. A mirror 19 is arranged.

  As shown in FIG. 2, the optical scanning unit 9 includes a scanning mirror element 20 such as a digital micromirror device (DMD) in which a plurality of minute mirrors 20a are two-dimensionally arranged. Each mirror 20a constituting the scanning mirror element 20 is selectively turned on / off by an operation command from an external control device 21. When each mirror 20a is placed in the ON state, it reflects the excitation light incident from the light source unit 6 side into the optical path toward the first objective lens 2, and the return light incident from the direction of the first objective lens 2 is reflected. The light is reflected in the optical path toward the image pickup means 7. On the other hand, when each mirror 20a is arranged in an off state, the excitation light and the return light are reflected in a direction deviating from these optical paths (in the direction of arrow C in FIG. 1).

  The scanning mirror element 20 constituting the optical scanning unit 9 is disposed at the first intermediate image position B by the condenser lens 14. Then, the first intermediate image of the white light source 10 is simultaneously received by the plurality of mirrors 20a and reflected in the direction of the first objective lens 2 by the mirror 20a that is turned on.

  As described above, the scanning mirror element 20 switches the on / off state of each mirror 20a in an arbitrary operation pattern by the control device 21. For example, FIG. 2A shows an operation pattern in which only a single mirror 20a indicated by diagonal lines X is turned on, and the mirror 20a that is turned on is switched according to the order indicated by the arrows in the figure. Accordingly, the sample A can be scanned with a single light spot as in the case where the light spot is scanned by swinging two galvanometer mirrors around two orthogonal axes.

  FIG. 2B shows an example of an operation pattern in which the mirrors 20a indicated by oblique lines X in all rows are turned on one by one from the square-arranged mirrors 20a. The mirrors 20a that are turned on in adjacent rows are shifted in the direction along the rows, and the on-state mirror 20a is sequentially switched over all rows as indicated by arrows in FIG. Dimensional scanning can be performed.

  FIG. 2C shows an example of an operation pattern when a plurality of adjacent mirrors 20a are simultaneously turned on. A large light spot can be scanned two-dimensionally on the sample A by moving the positions of the plurality of mirrors 20a indicated by oblique lines X, which are simultaneously turned on, as indicated by arrows.

  Moreover, each mirror 20a which comprises the scanning mirror element 20, and the fiber core 25 of each optical fiber which comprises the optical fiber bundle 4 are matched 1 to 1, as FIG. 3 shows. Thus, when only the single mirror 20a is turned on, the excitation light reflected by the mirror 20a is made incident on a predetermined single fiber core 25. On the other hand, the light emitted from the single fiber core 25 is incident and reflected only on the single mirror 20a associated with the fiber core 25 in advance.

  Reference numeral 22 in the drawing is disposed between the optical scanning unit 9 and the first objective lens 2, converts the excitation light reflected by the optical scanning unit 9 into parallel light and inputs the parallel light to the first objective lens 2. An imaging lens. Reference numeral 23 denotes a monitor that displays an image picked up by the image pickup means 7.

The operation of the optical scanning observation apparatus 1 according to the present embodiment configured as described above will be described.
In order to perform observation using the optical scanning observation apparatus 1 according to the present embodiment, an operator can freely move the measurement head 24 including the second objective lens 5 with respect to the sample A to perform appropriate observation. Place it in a position suitable for performing. Since the measurement head 24 is separated from the relatively large apparatus main body 3 and connected by the optical fiber bundle 4, the optical fiber bundle 4 can be freely bent so that the measurement head 24 can be disposed at an appropriate position and posture with respect to the sample A. it can.

  In this state, as shown in FIG. 2D, all the mirrors 20 a of the scanning mirror element 20 are turned on, and the excitation light emitted from the light source unit 6 is applied onto the scanning mirror element 20 by the condenser lens 14. The light is condensed and a first intermediate image is formed on the scanning mirror 20. Since all the mirrors 20a of the scanning mirror element 20 are in the on state, the first intermediate image is reflected by the scanning mirror element 20 as it is and is incident on the optical fiber bundle 4 through the imaging lens 22 and the first objective lens 2. It is done.

  Since each mirror 20a of the scanning mirror element 20 and each fiber core 25 of the optical fiber bundle 4 are in one-to-one correspondence, the excitation light propagates through all the fiber cores 25 and is sampled by the second objective lens 5. An image is formed on A. The fluorescence generated in the sample A is generated by the second objective lens 5, the optical fiber bundle 4, the first objective lens 2, the imaging lens 22, the scanning mirror element 20, the dichroic mirror 19, the condensing lens 18, the barrier filter 15, and the photometric filter. 16 and the condensing lens 17 are imaged by the imaging means 7.

  In this case, since all the mirrors 20a of the scanning mirror element 20 are turned on, no confocal effect is generated, and fluorescence from a focal position arranged at a predetermined depth position inside the sample A is detected. Although it is difficult to see only the image, a bright fluorescent image with a large amount of light can be obtained, and fluorescence emitted from a wide range in the depth direction is acquired by the imaging means 7, so that the operator can monitor the monitor 23. It is possible to easily find a desired observation target site while looking at the image, and to focus on the observation target site.

After the confirmation of the observation target region and the focus position alignment are completed, the operator switches to the measurement mode in which the control device 21 controls the scanning mirror element 20 with a predetermined operation pattern.
For example, when the operation pattern shown in FIG. 2A is performed, when excitation light emitted from the light source unit 6 is incident on the scanning mirror element 20 via the dichroic mirror 19, the excitation light is turned on. The light is reflected only by the single mirror 20a, and is incident on the end face of the corresponding fiber core 25 arranged on the one end face 4a of the optical fiber bundle 4 via the imaging lens 22 and the first objective lens 2. The excitation light propagated in the optical fiber bundle 4 is irradiated onto the sample A through the second objective lens 5 to generate fluorescence in the sample A.

  The fluorescence generated in the sample A returns to the scanning mirror element 20 through the second objective lens 5, the optical fiber bundle 4, the first objective lens 2 and the imaging lens 22. In the sample A, fluorescence is generated at various positions and is incident on the second objective lens 5, but forms intermediate images with the fiber core 25 on one end face 4 a of the optical fiber bundle 4 and each mirror 20 a of the scanning mirror element 20. Therefore, only the fluorescence emitted from the predetermined depth position of the sample A is imaged by the imaging means 7 by the confocal pinhole effect. As a result, as in the case of using a normal fixed confocal pinhole, it is possible to obtain a clear fluorescent image inside the sample extending to a predetermined depth position.

  According to the optical scanning observation apparatus 1 according to the present embodiment, the fiber core 25 of the optical fiber bundle 4 and the mirror 20a of the scanning mirror element 20 have a one-to-one correspondence, so the mirror 20a of the scanning mirror element 20 The pumping light reflected in the optical fiber bundle 4 can be incident on the fiber core 25 without being scattered by the clad portion 26 of the optical fiber bundle 4, and therefore, there is an advantage that the pumping light can be efficiently used without waste. .

  In addition, by switching the on / off state of each mirror 20a of the scanning mirror element 20 using the operation pattern shown in FIG. The sample A can be irradiated at the same time, and fluorescence from each position can be imaged simultaneously. Therefore, there is an advantage that rapid fluorescence observation can be performed.

Furthermore, the sample A can be scanned with a relatively large light spot by switching the on / off state of each mirror 20a of the scanning mirror element 20 by using the operation pattern shown in FIG. it can.
In this case, since the fluorescence generated in the vicinity of the site to be observed deviated from the focal position arranged inside the sample A can also pass through the one end face 4a of the optical fiber bundle 4 and the scanning mirror element 20, While the confocal effect is reduced, a bright image can be obtained by increasing the amount of fluorescence obtained in the imaging means 7. That is, it is effective for a purpose of performing observation with a bright fluorescent image even when the resolution is slightly lowered, or a purpose of acquiring a fluorescent image over a wide range in the depth direction.

  As described above, according to the optical scanning observation apparatus 1 according to the present embodiment, the on / off of each mirror 20a constituting the scanning mirror element 20 is controlled by an arbitrary operation pattern by the command signal from the control device 21. The resolution of the obtained fluorescent image can be freely changed. At this time, a bright image can be obtained by increasing the amount of fluorescence in exchange for a decrease in resolution, and observation over a wide range in the depth direction can be performed.

  In the optical scanning observation apparatus 1 according to the present embodiment, each mirror 20a of the scanning mirror element 20 and each fiber core 25 of the optical fiber bundle 4 are associated one-to-one. Thus, the correspondence may be one-to-many or many-to-one. If one-to-many (for example, one-to-four, one-to-nine, etc.) and many-to-one (for example, four-to-one, nine-body one, etc.) Although it will be reduced, a bright fluorescent image can be obtained by increasing the amount of fluorescence obtained. Further, by keeping the ratio between the mirror 20a and the fiber core 25 associated with each other constant for all the mirrors 20a, a uniform fluorescent image can be obtained without causing bright and dark in the fluorescent image.

  In the optical scanning observation apparatus 1 according to the present embodiment, a combination of the white light source 10 such as a xenon lamp or a mercury lamp and the excitation filter 11 is used as the light source unit 6. A laser light source may be employed.

Next, an optical 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 optical scanning observation apparatus 1 according to the first embodiment described above, and the description will be simplified.

The optical scanning observation apparatus 30 according to the present embodiment is the first in that the light source unit 31 includes a laser light source 32 and a cylindrical lens 33 and generates strip-shaped parallel light, and the structure of the optical scanning unit 34 is different. This is different from the optical scanning observation apparatus 1 according to the embodiment.
The light source unit 31 includes a collimating lens 35, and converts the laser light emitted from the laser light source 32 into parallel light.

  The optical scanning unit 34 reflects the belt-shaped laser beam emitted from the light source unit 31 at the first intermediate image position B of the condenser lens 36, and the laser beam reflected by the scanning mirror device 37. A collimating lens 38 for converting the parallel light into a band, a galvano mirror 39 for reflecting the parallel light emitted from the collimating lens 38, and a laser beam reflected by the galvano mirror 39 to collect the third intermediate image position. And a pupil projection lens 40 for forming a third intermediate image on D.

As shown in FIG. 5, the scanning mirror element 37 includes a plurality of mirrors 37a arranged in a line at a position where the strip-shaped laser light L is simultaneously incident, and the on / off states of the mirrors 37a are sequentially changed in the arrangement direction. By switching, the laser beam L can be scanned in one direction.
The galvanometer mirror 39 is rotated by a motor 41 around an axis line arranged in the scanning plane of the laser beam L by the scanning mirror element 37. As a result, the laser light L scanned in one direction by the scanning mirror element 37 can be further scanned by the galvano mirror 39 in a direction intersecting therewith.
That is, also in this embodiment, the laser beam is scanned in the two-dimensional direction by the optical scanning unit 34 and is incident on the one end surface 4 a of the optical fiber bundle 4.

  According to the optical scanning observation apparatus 30 according to the present embodiment configured as described above, the sample can be formed with a simple configuration by combining the scanning mirror element 37 in which the mirrors are arranged in one row and the uniaxial galvanometer mirror 39. A can be scanned two-dimensionally. In this case, compared to the case where scanning is performed by two galvanometer mirrors, the strip-shaped parallel light is incident on the scanning mirror element 37, so that the laser light L reflected by each mirror 37a constituting the scanning mirror element 37 is The amount of light can be increased. Therefore, a bright fluorescent image can be obtained by allowing a high amount of laser light to enter the sample A.

  In the optical scanning observation device 30 according to the present embodiment, the scanning mirror element 37 is configured by arranging the mirrors 37a in one row. Instead, as shown in FIG. You may employ | adopt what arranged more than row | line | column (FIG. 6 is 4 rows, for example). Then, by selecting arbitrarily the mirrors 37a to be simultaneously turned on (for example, selecting four as shown by the hatched lines X in FIG. 6), the same as the optical scanning observation apparatus 1 according to the first embodiment. The effect can be achieved.

1 is an overall configuration diagram showing an optical scanning observation apparatus according to a first embodiment of the present invention. It is a figure which shows the example of an operation pattern of the scanning mirror element of the optical scanning observation apparatus of FIG. It is a schematic diagram which shows a response | compatibility with the scanning mirror element of this invention, and the fiber core of an optical fiber bundle. It is a whole block diagram which shows the optical scanning type observation apparatus which concerns on the 2nd Embodiment of this invention. It is a schematic diagram which shows the scanning mirror element of the optical scanning observation apparatus of FIG. It is a schematic diagram which shows the modification of FIG.

Explanation of symbols

A Sample B Second intermediate image position 1,30 Optical scanning observation device 2 First objective lens 4 Optical fiber bundle 4a One end face 4b Other end face 5 Second objective lens 6,31 Light source section 7 Imaging means 14, 36 Condensing lens 20, 37 Scanning mirror element 20a, 37a Mirror 21 Controller 22 Imaging lens 25 Fiber core 39 Galvano mirror

Claims (4)

A light source unit, and a condensing lens that forms a first intermediate image of excitation light emitted from the light source unit in a line ,
An imaging lens for condensing the intermediate image;
A first objective lens that forms a second intermediate image of the excitation light condensed by the imaging lens;
An optical fiber bundle having one end face in the vicinity of the second intermediate image position;
A second objective lens that is disposed on the other end surface side of the optical fiber bundle and forms an image of light emitted from the other end surface of the optical fiber bundle on a sample;
The second objective lens, the optical fiber bundle, the first objective lens, and imaging means for imaging the return light returning through the imaging lens,
Wherein the first intermediate image position, at least one row sequence and comprising a scanning mirror elements selectively a plurality of mirrors that can be turned on and off with receiving the first intermediate image simultaneously are arranged,
An optical scanning observation apparatus including a galvanometer mirror that scans light reflected by each mirror constituting the scanning mirror element in a direction crossing the column direction of the mirror .   The optical scanning observation apparatus according to claim 1, further comprising a control device that controls an operation pattern of each mirror constituting the scanning mirror element.   3. The optical scanning observation apparatus according to claim 1, wherein each mirror constituting the scanning mirror element and each fiber core constituting the optical fiber bundle are arranged in a one-to-one correspondence. 4.   The light according to claim 1 or 2, wherein each mirror constituting the scanning mirror element and each fiber core constituting the optical fiber bundle are arranged so as to correspond one-to-many or many-to-one. Scanning observation device.

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