JP2009192721A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscope capable of making precise sectioning even when exchanging the objective lens and to obtain correct confocal images of desired sectioning resolution after acquiring images of samples. <P>SOLUTION: The confocal microscope has a scanning means 16 to scan the light of a light source 5, an objective lens 13 to focus the light passing through the scanning means 16 on a sample 3, a splitter 19 to divide the path of the observation light, a first pinhole 20 which can change its diameter and is disposed on one of the light path divided by the splitter 19 at the position substantially conjugated to the focal point of the objective lens 13, a second pinhole 22 which has a diameter different from that of the first pinhole 20 and is disposed on the other one of the light path divided by the splitter 19 at the position substantially conjugated to the focal point of the objective lens 13, and an operation means 37 to form, based on the data of the two confocal images obtained from the two light beams different in sectioning resolution and passing through these pinholes 20, 22, a confocal image different in sectioning resolution from the confocal images. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a confocal microscope.

Conventionally, confocal microscopes that perform observation by limiting (sectioning) the observation target to a thin layer near the focal plane of the specimen by placing a pinhole at a position conjugate with the focal position of the objective lens are known, In recent years, it has been proposed to change the thickness of this layer to be observed, so-called sectioning resolution.
In such a background, the present applicant separates and detects the observation light from the specimen into two light beams having different sectioning resolutions by using a light separation means having two pinholes having different aperture diameters, thereby detecting two confocal images. A confocal microscope configured to create a confocal image having a desired sectioning resolution using these image data, that is, a confocal microscope capable of observing a confocal image in which the sectioning resolution is changed after image acquisition of the specimen (For example, refer to Patent Document 1).
JP 2005-274591 A

  Here, in the conventional confocal microscope as described above, the optimal aperture diameter for the objective lens of the pinhole provided in the light separating means varies depending on the magnification and numerical aperture of the objective lens to be used. However, in the conventional confocal microscope, since the aperture diameter of the pinhole of the light separating member is fixed, when switching the objective lens, the accuracy of sectioning is reduced, and the sectioning resolution is changed. In addition, there is a problem that it becomes difficult to obtain an accurate confocal image of the specimen.

  Therefore, the present invention has been made in view of the above problems, and it is possible to perform sectioning with high accuracy even when the objective lens is switched, and to obtain an accurate confocal image having a desired sectioning resolution after obtaining a sample image. An object of the present invention is to provide a confocal microscope.

In order to solve the above problems, the present invention
Scanning means for scanning light from the light source;
An objective lens for condensing the light passing through the scanning means on the specimen;
Optical path dividing means for dividing the optical path of the observation light from the specimen;
A first pinhole means capable of changing the aperture diameter disposed on one of the optical paths divided by the optical path dividing means at a position substantially conjugate with the focal point of the objective lens; and the first pinhole means and the opening Second pinhole means disposed on the other optical path having a different aperture and substantially conjugate with the focal point of the objective lens and divided by the optical path dividing means;
Based on the data of two confocal images obtained from two light beams having different sectioning resolutions that have passed through the first pinhole unit and the second pinhole unit, the confocal image and the confocal image having different sectioning resolutions are obtained. A computing means to create;
A confocal microscope is provided.

  According to the present invention, it is possible to provide a confocal microscope that can accurately perform sectioning even when the objective lens is switched, and can obtain an accurate confocal image having a desired sectioning resolution after obtaining an image of the specimen. Can do.

Hereinafter, a confocal microscope according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.
Fig.1 (a) is a schematic block diagram which shows the structure of the confocal microscope which concerns on embodiment of this invention.
As shown in FIG. 1A, the microscope apparatus 1 according to the present embodiment supplies illumination light to the inverted microscope 2, the confocal observation system 4 for performing the confocal observation of the specimen 3, and the confocal observation system 4. Laser unit 5, two light detection units 6 and 7 for detecting observation light (fluorescence) of the specimen 3 obtained by the confocal observation system 4, and a controller 8.

  The inverted microscope 2 includes a transmission illumination device 9, a stage 10 on which the sample 3 is placed, an objective lens turret 11, and an imaging lens 12 in order from above. The objective lens turret 11 includes a plurality of objective lenses 13, and a desired objective lens 13 can be switched and disposed in the optical path by rotating the objective lens 13. The inverted microscope 2 includes an optical system (not shown) for guiding the observation light from the specimen 3 to the eyepiece observation unit 14.

  The confocal observation system 4 is directly attached to the main body 2a of the inverted microscope 2, and in order from the imaging lens 12 side of the inverted microscope 2, a scanner lens 15, a galvano scanner 16 that scans light two-dimensionally, a dichroic. It has a mirror 17, a condenser lens 18, a half mirror 19, and a first pinhole turret 20. A second pinhole turret 22 is provided on the reflection optical path of the half mirror 19 via a total reflection mirror 21. A collimating lens 23 is provided on the reflected light path of the dichroic mirror 17.

  The first pinhole turret 20 is disposed at a position conjugate with the focal position of the objective lens 13, and a plurality of pinholes 20a having different opening diameters are provided along the circumference as shown in FIG. The desired turret member can be rotated and operated to switch the desired pinhole 20a into the optical path. Specifically, in the present embodiment, the first pinhole turret 20 is provided with pinholes 20a having diameters of 30, 100, 120, 150, 180, 200, 250, and 300 μm. With such a configuration of the first pinhole turret 20, a pinhole 20a having an optimum opening diameter can be selected and arranged in the optical path according to the objective lens 13 to be used.

  The second pinhole turret 22 has the same configuration as the first pinhole turret 20, and in this embodiment, includes a pinhole 22a having a diameter larger than that of the pinhole 20a provided in the first pinhole turret 20. ing. Specifically, the second pinhole turret 22 is provided with pinholes 22a having diameters of 200, 250, 300, 350, 400, 450, 500, and 550 μm. With such a configuration of the second pinhole turret 22, a pinhole 22a having a desired opening diameter can be selected and arranged in the optical path in accordance with the pinhole 20a selected by the first pinhole turret 20 described above. .

  The laser unit 5 is connected to the confocal observation system 4 through an optical fiber 5a, and has a condenser lens 24, a dichroic mirror 25, and a total reflection mirror 26 in order from the collimating lens 23 side of the confocal observation system 4. On the reflected light paths of these mirrors 25 and 26, shutter devices 27a and 28a and laser light sources 27b and 28b for emitting laser beams having different wavelengths are provided. Note that the shutter devices 27a and 28a also include an acoustooptic device for adjusting the power of the laser light.

The light detection unit 6 is connected to the first pinhole turret 20 of the confocal observation system 4 through an optical fiber 6a, and in order from the input port side of the optical fiber 6a, a lens 30, a dichroic mirror 31, and the dichroic. A dichroic mirror 32 having a wavelength characteristic different from that of the mirror 31 is provided. Barrier filters 33a, 34a, 35a, condenser lenses 33b, 34b, 35b, and PMTs 33c, 34c, 35c are provided on the transmission optical path and the reflection optical path of each dichroic mirror 31, 32, respectively.
The light detection unit 7 has the same configuration as that of the light detection unit 6, and is connected to the second pinhole turret 22 of the confocal observation system 4 through an optical fiber 7a.

  The controller 8 is connected to the computer 37 and controls each part of the confocal microscope 1 based on instructions from the computer 37. Specifically, in order to control the inverted microscope 2, the galvano scanner 16 of the confocal observation system 4, the laser unit 5, and the light detection units 6 and 7, the controller 8 corresponds to the control units 8 a, 8 b, and 8 c respectively. , 8d. Note that a monitor 38 for displaying an observation image or the like of the specimen 3 is also connected to the computer 37.

Next, confocal observation of the specimen 3 at a desired sectioning resolution performed using the confocal microscope 1 having the above-described configuration will be described.
First, an observer of the confocal microscope 1 selects a pinhole 20a having an optimum aperture diameter from the first pinhole turret 20 according to the magnification and numerical aperture of the objective lens 13 to be used, and arranges it in the optical path. A pinhole 22a having a desired opening diameter is selected from the second pinhole turret 22 in accordance with the selected pinhole 20a and arranged in the optical path. Specifically, assuming that the opening diameter of the pinhole 20a is a, the opening diameter b of the pinhole 22a is selected to satisfy the relationship of b = 2a, 3a.

  Under this premise, in the confocal microscope 1 according to the present embodiment, the light emitted from the laser light source 27 b in the laser unit 5 is reflected by the dichroic mirror 25 through the shutter device 27 a and enters the condenser lens 24. Further, the light emitted from the other laser light source 28 b is reflected by the total reflection mirror 26 through the shutter device 28 a, further passes through the dichroic mirror 25, and enters the condenser lens 24. As a result, the laser beams of the laser light sources 27b and 28b are collectively collected by the condenser lens 24 on the end surface of the optical fiber 5a, and input to the confocal observation system 4 as illumination light through the optical fiber 5a.

  The illumination light input to the confocal observation system 4 is reflected by the dichroic mirror 17 through the collimating lens 23 and input to the inverted microscope 2 through the galvano scanner 16 and the scanner lens 15. This illumination light is irradiated to the specimen 3 on the stage 10 through the imaging lens 12 and the objective lens 13 in this order in the main body 2 a of the inverted microscope 2.

  As a result, the observation light emitted from the specimen 3 is input to the confocal observation system 4 through the objective lens 13 and the imaging lens 12 again in this order. In the confocal observation system 4, this observation light again passes through the scanner lens 15 and the galvano scanner 16, passes through the dichroic mirror 17, and further passes through the condenser lens 18, and is guided to the half mirror 19. Divided equally into reflected light. The transmitted light from the half mirror 19 is condensed on the pinhole 20a of the first pinhole turret 20, and the light that has passed through the pinhole 20a is input to the light detection unit 7 via the optical fiber 7a. . On the other hand, the reflected light from the half mirror 19 is reflected by the total reflection mirror 21 and focused on the pinhole 22a of the second pinhole turret 22, and the light passing through the pinhole 22a passes through the optical fiber 6a. Then, the light is input to the light detection unit 6.

  Next, the observation light (observation light from the first pinhole turret 20) input to the light detection unit 7 is separated into three wavelength components by the two dichroic mirrors 31 and 32 after passing through the lens 31. After passing through the corresponding barrier filters 33a, 34a, and 35a and the condenser lenses 33b, 34b, and 35b in order, they are detected by the PMTs 33c, 34c, and 35c. Since the illumination light applied to the specimen 3 is two-dimensionally scanned by the galvano scanner 16 as described above, the observation light is detected over the entire observation area of the specimen 3 in each PMT 33c, 34c, and 35c. It becomes.

Thereby, the computer 37 acquires the detection signal for each wavelength component of the observation light from each of the PMTs 33c, 34c, and 35c of the light detection unit 6 through the controller 8, and creates two-dimensional image data of the sample 3 based on the detection signal. And stored in a memory (not shown) provided in the computer 37.
The two-dimensional image data of the specimen 3 obtained here passes through a pinhole 20a having an optimum opening diameter with respect to the objective lens 13 among the small-diameter pinholes 20a provided in the first pinhole turret 20. Since it is based on the observed light, it has information on a thin layer in the vicinity of the focus surface in the specimen 3, that is, information with high sectioning resolution and accurate information with high sectioning accuracy.

On the other hand, the observation light (observation light from the second pinhole turret 22) input to the light detection unit 6 is detected by the light detection unit 6 in the same manner as the observation light input to the light detection unit 7 described above. Two-dimensional image data of the sample 3 for each wavelength component is created by the computer 37 and stored in the memory.
The two-dimensional image data of the specimen 3 obtained here is based on the observation light that has passed through the large-diameter pinhole 22a of the second pinhole turret 22, so that the thick layer around the focal plane in the specimen 3 is used. Information, that is, information with low sectioning resolution.

As described above, the computer 37 that obtains the two-dimensional image data with the high sectioning resolution and the two-dimensional image data with the low sectioning resolution for each wavelength component performs the calculation on these to obtain the sectioning resolution specified by the observer. Are generated for each wavelength component and displayed on the monitor 38 as an observation image. The calculation is D = Dbα + (1−α) Da, where Da is a two-dimensional image data having a high sectioning resolution and Db is a two-dimensional image data having a low sectioning resolution (α is a weighting factor, and −1 ≦ α ≦ + 1).
In this way, the observer can observe the specimen 3 confocally with a desired sectioning resolution for each wavelength component.
The sectioning resolution can be changed by the observer through an input unit (not shown) provided in the computer 38 regardless of before and after the acquisition of the two-dimensional image data of the specimen 3.

  With the above configuration, the confocal microscope 1 according to the present embodiment can change the sectioning resolution, and can obtain a confocal image having a desired sectioning resolution after the image of the specimen 3 is acquired. Further, by selecting and using the pinhole 20a having the optimum aperture diameter for the objective lens 13 to be used as described above from the first pinhole turret 20, it becomes possible to perform high-precision sectioning. By using the accurate two-dimensional image data for the above-described calculation, an accurate confocal image having a desired sectioning resolution can be obtained.

  Furthermore, the confocal microscope 1 according to the present embodiment can change the resolution of the two-dimensional image data having the low sectioning resolution by switching the pinhole 22a of the second pinhole turret 22, and this can be used for the above-described calculation. By using it, it becomes possible to change the brightness of the confocal image having a desired sectioning resolution. For example, in the present embodiment, by switching the pinhole 22a of the second pinhole turret 22 to one having a large diameter, it is possible to obtain a brighter confocal image with a desired sectioning resolution.

  As described above, according to the present embodiment, it is possible to perform sectioning with high accuracy even when the objective lens 13 is switched, and it is possible to obtain an accurate confocal image having a desired sectioning resolution after acquiring the image of the specimen 3. The focusing microscope 1 can be realized. Moreover, in this embodiment, since it is the structure provided with the 1st pinhole turret 20 and the 2nd pinhole turret 22 of a simple structure compared with the light separation means in the conventional confocal microscope mentioned above, manufacture is easy. Can also be realized.

  In addition, the structure of the 1st pinhole turret 20 in this embodiment is not restricted to the above-mentioned thing. For example, a pinhole having an optimum aperture diameter determined based on the magnification and numerical aperture of the objective lens 13 may be provided for the plurality of objective lenses 13 that are used by switching in the confocal microscope 1. This makes it possible to maximize the effects of the present invention. Further, for example, instead of the first pinhole turret 20, a pinhole member having a variable aperture diameter may be provided at a position conjugate with the focal position of the objective lens 13, thereby achieving the same effect as the present invention. Can play. This also applies to the second pinhole turret 22.

(A) is a schematic block diagram which shows the structure of the confocal microscope which concerns on embodiment of this invention, (b) is a figure which shows the structure of a 1st pinhole turret and a 2nd pinhole turret.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Confocal microscope 2 Inverted microscope 3 Sample 4 Confocal observation system 5 Laser unit 6, 7 Photodetection unit 8 Controller 13 Objective lens 16 Galvano scanner 19 Half mirror 20 1st pinhole turret 22 2nd pinhole turret 37 Computer

Claims (5)

Scanning means for scanning light from the light source;
An objective lens for condensing the light passing through the scanning means on the specimen;
Optical path dividing means for dividing the optical path of the observation light from the specimen;
A first pinhole means capable of changing the aperture diameter disposed on one of the optical paths divided by the optical path dividing means at a position substantially conjugate with the focal point of the objective lens; and the first pinhole means and the opening Second pinhole means disposed on the other optical path having a different aperture and substantially conjugate with the focal point of the objective lens and divided by the optical path dividing means;
Based on the data of two confocal images obtained from two light fluxes having different sectioning resolutions that have passed through the first pinhole unit and the second pinhole unit, the confocal image and the confocal image having different sectioning resolutions are obtained. A computing means to create;
A confocal microscope characterized by comprising:   The confocal microscope according to claim 1, wherein the aperture diameter of the first pinhole means is changed based on a magnification and a numerical aperture of the objective lens.   The confocal microscope according to claim 1 or 2, wherein the second pinhole means is capable of changing an opening diameter.   The confocal microscope according to any one of claims 1 to 3, wherein the first pinhole means includes a turret member that can switch a plurality of pinholes having different opening diameters.   5. The confocal microscope according to claim 1, wherein the second pinhole means is formed of a turret member that can switch a plurality of pinholes having different opening diameters.

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