WO2006085480A1

WO2006085480A1 - Cofocal microscope

Info

Publication number: WO2006085480A1
Application number: PCT/JP2006/301826
Authority: WO
Inventors: Susumu Terakawa; Takashi Sakurai; Yoshihiko Wakazono; Seiji Yamamoto
Original assignee: National University Corporation, Hamamatsu University School Of Medicine
Priority date: 2005-02-09
Filing date: 2006-02-03
Publication date: 2006-08-17
Also published as: JP2006220818A

Abstract

A Nipkow system cofocal microscope in which a region of a sample can be illuminated selectively and observed using a DMD. Illumination light from a light source (20) passes through a condenser lens (1) and a rotary filter (14) and impinges on a DMD (2) and only the light subjected to on-control by the DMD (2) and illuminating a micro mirror region impinges on the microlens (5) of a Nipkow disk assembly. The light passed through the microlens (5) is converged by a pinhole (6) to form an image in a selected region of a sample (10) by an objective lens (8). Reflected light, e.g. fluorescence, from the selected region of a sample (10) retrogresses along the incident optical path and is reflected by a dichroic mirror (7) before an image is formed on a two-dimensional photodetector by an image forming lens (11).

Description

Specification

Confocal microscope

Technical field

[0001] The present invention relates to a confocal microscope using a bipco plate.

Background art

[0002] A confocal microscope that can obtain an optically cut image is widely used in bioresearch. To achieve this, a laser beam is squeezed into a dot shape, and the image at this point is rotated in a fan shape. Swing and laser scanning with a bright spot formed on the specimen, and a two-puck plate type where a disk with a small hole (pinhole) is rotated instead of a mirror and is moved by a pinhole image formed on the specimen. There is. The fluorescent material of the specimen is excited by bright spot scanning, and the resulting fluorescent light can be collected through a pinhole at the conjugate position and observed as an image. The micro-lens-Pukou plate, which has improved the light utilization efficiency through the combination of microlenses, is brighter and faster scanning, and its use is relatively widespread. For example, the present inventors have proposed that the Puku disc assembly is used in Patent Document 1 and Patent Document 2.

[0003] However, the disadvantage of this method is that illumination light cannot be applied to only a specified region of the field of view and only that region is observed, and illumination and observation are always performed on the entire field of view. . This may be inconvenient for observation of fluorescently stained cells. That is, fluorescence observation involves a phenomenon called fading of the observation object, and the object is changed to an invisible one depending on the observation time and the applied light intensity.

If only cells of interest in a single field of view can be selectively illuminated and observed, those not targeted will not be exposed to light and can be observed sequentially.

[0004] On the other hand, there is a technique using DMD that can be realized by the former laser system.

This includes Patent Documents 3, 4 and 5.

The invention described in Patent Document 3 discloses a technique in which light emitted from a light source is reflected by an on-element of a DMD and focused on an object plane. The fluorescence excited in the object plane returns to the DMD through the same optical path, and is reflected by the semitransparent mirror to reach one camera. Out of focus The generated light is reflected off the mirror by the off-element and reaches the other camera.

The invention of an optical apparatus and method for imaging in a scanning microscope and a scanning microscope described in Patent Document 4 is a confocal in which laser light is reflected by a micromirror and applied to a specimen, and the reflected light is detected. It is a scanning microscope.

[0005] Patent Document 5 was proposed by the present inventor and is a confocal scanning microscope using an epi-illumination type and a transmission type DMD. The DMD is used to determine the size of the bright spot, the scanning speed, and the scanning pattern. A confocal microscope that can be freely changed is realized.

In any of the above Patent Documents 3, 4 and 5, the scanning area of the specimen can be selected, but the light utilization efficiency is inferior to that of the microlens-Pukou plate type, resulting in a light image. Patent Document 6 discloses a device having a DMD as a device for controlling an irradiation region using a fluorescent microscope instead of a microscope.

The invention described in Patent Document 6 relates to a fluorescence microscope. In FIG. 1, the illumination light emitted from the light source 23 is collected by the collector lens 22 and selectively reflected by the digital micromirror 201 to cause the digital micromirror to be reflected. Use as a field stop to enable partial illumination of the specimen to prevent fading, but a description indicating or suggesting its application to a confocal microscope with a microlens-Pukou plate There is no.

Patent Document 1: Japanese Patent Application No. 2003-187142

Patent Document 2: Patent No. 3099063

Patent Document 3: Japanese Patent Laid-Open No. 11-194275

Patent Document 4: Japanese Patent Laid-Open No. 2002-131649

Patent Document 5: Japanese Patent Application No. 2003-186768

Patent Document 6: Japanese Unexamined Patent Publication No. 2003-107361

Disclosure of the invention

Problems to be solved by the invention

[0006] The present invention solves the above-mentioned drawbacks of the above-described two-puko plate type confocal microscope, and its object is to improve the light utilization efficiency in the two-pcho type confocal microscope. An object of the present invention is to provide a confocal microscope capable of selectively illuminating and observing an area of a specimen using DMD.

Means for solving the problem

In order to achieve the above object, claim 1 of the present invention includes a light source device and a plurality of micromirrors arranged two-dimensionally, and the light from the light source device is punctuated by the selected micromirrors. From DM D, which converts the illumination light into a parallel light beam according to the selected set of micromirrors, and the 1st 2 Puk board with many pinholes and the 2nd Pukou board with many microlenses The parallel light beam from the DMD is converged by the micro lens of the second Puk plate and imaged in the pin hole of the first Puk plate-Puk plate assembly, and the pin of the first Puk plate A light beam imaged in a hole is imaged at a corresponding position in the field of view, and is disposed between a confocal optical system that reverses fluorescence from the field of view, a two-dimensional light detection means, and the first and second two-ply plates. Color-splitting optical element and color-splitting optical element An output optical system comprising a lens system that focuses reflected light from a child on the light detection means, and a control device that selects and controls on-off control of a number of micro mirrors of the DMD, It is characterized in that it is configured so that a part of the light is selected and illumination is given.

A second aspect of the present invention is characterized in that, in the first aspect of the present invention, the two-piece plate assembly is rotated at a high speed to form an image on the light detecting means.

A third aspect of the present invention is characterized in that, in the first aspect of the present invention, a fluorescent specimen is placed in the visual field.

The invention's effect

[0008] According to the configuration described above, it is possible to observe only the irradiated region by irradiating only the arbitrarily designated region of the field of view with the light with improved light use efficiency of the light source.

If the wavelength of the laser is selected, the light release region of the caged compound and the intensity of the release light, and the observation region are arbitrarily selected by DMD, the sample is observed by the light of the caged compound while observing the specimen with a two-ply plate confocal microscope. It can also be applied to release and can be used for a wide range of applications of caged reagents. For example, there is a chemical stimulus of a living specimen such as a cell, and the reaction of the resulting cell can be captured as it is by fluorescence observation with a confocal microscope. Fig. 7 shows an example of a fluorescence image when each region of a living specimen such as a cell is chemically stimulated. When searching for the localization of a specific receptor in a certain cell, it is possible to observe the intracellular response by sequentially stimulating light release in multiple microregions.

In addition, the setting of the illumination area (or ROI) by DMD is not limited to monotonous figures such as circles and squares, and complex pictures and characters such as outlines of cell morphology are possible, and characters can be printed on cells. For example, it can take the form of printing limited to a certain limited fault plane.

A light source other than a laser can also be used. If a light source that mixes several wavelengths of light (such as white light) is used, the tone adjustment function of the DMD is synchronized with the color specification of the filter that can be switched at high speed, and the color of each region Can also be projected.

Brief Description of Drawings

FIG. 1 is a block diagram showing an outline of a confocal microscope according to the present invention.

FIG. 2 is a diagram showing an embodiment of a confocal microscope according to the present invention.

FIG. 3 is a diagram showing an example of a rotary filter.

FIG. 4 is a diagram for explaining the ROI of DMD.

FIG. 5 is a diagram for explaining an example of a two-ply plate assembly.

FIG. 6 is a diagram for explaining the generation of fluorescence in a selected region of a specimen.

FIG. 7 is a diagram showing an example of a fluorescence image when each region of a living specimen such as a cell is chemically stimulated in order.

Explanation of symbols

[0010] 1 condenser lens

2 DMD (digital 'micromirror' device)

3 Micro mirror group (ON state)

4 With microlens-Pukou board

5 Micro lens

6 Pinhonore

7 color split mirror (dichroic mirror)

8 Objective lens 9 Cover glass

10 Sample area to be a confocal image

11 Imaging lens

12 Two-dimensional photodetector (camera)

13 Light trap

14 Rotary filter

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings and the like.

The confocal microscope is equipped with a confocal optical system, two-ply plate assembly, output optical system, DMD, and illumination optical system. Light having the power of a light source device enters the illumination optical system.

The camera device detects the optical signal from the output optical system and outputs it to the monitor. The control device composed of CPU, memory circuit, control program, etc. functions as a DMD controller that controls on / off of the micromirrors in the selected area of the DMD and a rotation controller that controls the rotation of the two-ply plate ^ a. It is equipped with.

When unifying the fluorescence intensity in multiple areas or tracking a moving sample, the output signal of the two-dimensional photodetector is fed back to the control device including the DMD control unit, and the control device uses the excitation light intensity ( It is configured to automatically adjust the ROI using the tone adjustment function of DMD).

When observing multiple areas with different excitation light using a white light source, the ROI, excitation light filter (provided in the illumination optical system, etc.), and dichroic mirror (including absorption filter, if any) are synchronized. In such a case, the control device including the DMD control unit is configured to simultaneously control the excitation filter and the dichroic mirror (including an absorption filter, if any).

The ROI of the sample placed in the field of view can be selected and input to the monitor by inputting the operating force.

[0012] The light of the light source device power is converted into a predetermined wavelength band by the illumination optical system and guided to the DMD. About the illumination range of the DMD ROI is selected under the control of the DMD control unit. On / off control is performed. The light from the on-controlled micro mirror group becomes a parallel light beam corresponding to the micro mirror group, and the parallel light beam is incident on the micro lens corresponding to the Puko plate assembly and converged by the micro lens. With a pinhole-Forms an image with a pinhole on a Pukow plate. The light focused on the pinhole is focused on a selected area of the specimen by the confocal optical system. Fluorescence and scattered light generated in the selected area of the specimen returns to the confocal optical system and is guided by the output optical system to the two-dimensional photodetector (camera device), where it is processed for image display and displayed on the monitor. .

By rotating the Pukko board assembly at a high speed by the rotation control unit, a predetermined number of specimen images can be obtained per second.

The condensing lens 1 and the rotary filter 14 constitute an illumination optical system. -Pukou plate assembly has a number of spiral pinholes 6 -Pukou plate 4a and a number of microlenses 5 provided for each pinhole -Pukou plate 4b is connected by rotating shaft 15 The The confocal optical system is composed of a plurality of microlenses 5-a Pukko plate 4b, a large number of pinholes 6-a Pukko plate 4a, and an objective lens 6 that forms an image at a conjugate position with respect to the pinhole position. The output optical system is composed of a dichroic mirror 7 and an imaging lens 11 inserted on the optical paths of the Pukko plates 4a and 4b. A specimen is mounted on the cover glass 9, and the specimen is a position including a conjugate position with respect to the pinhole 6 in the field of view of the microscope.

As the light source 20, a laser beam having a predetermined wavelength is used, and a light source in which several wavelengths are mixed is used. In this embodiment, a white light lamp is used, and the ROI can be projected in each color. Fig. 3 shows an example of a rotary filter inserted in the illumination optical system. A filter 14a in the red wavelength region, a filter 14b in the green wavelength region 14b, and a filter 14c in the blue wavelength region are arranged on the circumference, and are rotated by the rotating shaft 16 and projected by changing the color for each ROI. It is out.

The illumination light from the light source 20 is converted into a parallel light beam by the lens 1, passes through the rotary filter 14, and irradiates each micromirror of the DMD 2. Figure 4 shows the case where the specified ROD of the DMD is on-controlled. DMD2 25 is the irradiable area, and the area under the control of the DMD controller (R ) 2a and region (R) 2b micro mirrors are selected and turned on.

1 2

The other area of the irradiation area 25 is an area where the micromirror is off. Each region of the irradiation region 25 can be selected in turn by the DMD control unit. The selected area (R), (R) is a rectangular area, but its shape and size can be set freely.

1 2

The

[0016] On-controlled micromirror region (R) and reflected light from region (R) are parallel light fluxes

1 2

-Is incident on the corresponding microlenses 5 of the Pukou plate 4b.

On the other hand, the light in the irradiation region 25 other than the region (R) and the region (R) is reflected by the minute mirror in the region.

1 2

When the light is turned off, the light is guided in a direction other than the direction of the Pukko plate 4b and absorbed by the optical trap 13. The light incident on the corresponding microlens 5 is imaged on the corresponding pinhole 6. The two-Pukou plate configuration increases the efficiency of the incident light and reduces the noise light reflected on the plate surface other than the pinhole.

FIG. 5 shows an example of a two-ply plate assembly.

-The pinholes 6 of the Pukko plate 4a are arranged in a spiral at a predetermined interval, and a large number of these spiral pinhole groups are provided on the entire disc. -A large number of microlenses 5 are arranged on the Pukou plate 4b for each pinhole. -The specimen image can be displayed on the monitor by rotating the Pukou board assembly by the rotation controller. For example, the number of display frames per second can be changed by appropriately controlling the number of spiral pinhole groups and the rotational speed (minute speed) of the two-ply plate. For example:-By forming a Pukko board at several thousand rpm and about 10 spiral pinhole groups, one frame can be displayed at several mmS.

The light that has passed through the pinhole 6 is imaged at a selected position of the sample 10 by the objective lens 8.

Fig. 6 shows the selected position force fluorescence of the specimen.

DMD region (R) force light 26a is focused at the position where the front force of sample 10 is incident by z

1 1

Tie. Vertical and horizontal direction is area (R) y

Position X corresponding to 1,

1 Forms an image on 1. In addition, the light 26b from the DMD region (R) focuses on the position where the frontal force of the specimen 10 is incident by _Z , and the vertical and horizontal directions.

twenty one

Direction is region (R)

The position X, y corresponding to 2

2 Focus on 2

For example, fluorescence 27 is generated by the substance existing at the imaging position of the specimen 10, and the fluorescence 27 returns to the confocal optical system and is guided to the imaging lens 11 by the dichroic mirror 7 that reflects the fluorescence wavelength band. It is. Fluorescence forms an image on the two-dimensional photodetector 12 by the imaging lens 11, and the position of the specimen 10 x

1

, z and images X, y, z are detected.

1 1 2 2 1

The two-dimensional photodetector 12 performs photoelectric conversion, and this electric signal is processed into a predetermined signal system in the camera device and then displayed on a monitor.

In the above embodiment, an example in which a dichroic mirror is used for the output optical system has been described. However, other optical elements may be used as long as they are optical elements that can separate the output (fluorescence) wavelength band. . An absorption filter may be inserted into the dichroic mirror, the reflected light from the dichroic mirror may be guided to the absorption filter, and the transmitted light from the absorption filter may be imaged on the light detection means. Further, a force or wavelength range required for the light source device may be generated without inserting a force rotation filter in which an example is shown in which a rotary filter is inserted to make the irradiation light have an RGB wavelength range.

Force that can generate diffracted light due to the periodic surface structure of DMD In that case, force that uses only the 0th-order light of diffracted light, force that uses light that has passed through a diffuser or an incoherencer, etc. Alternatively, the present invention can be implemented by using a light source with low coherence (xenon lamp, SLD, femtosecond laser, etc.).

Furthermore, a specific example in which white light is used for the light source device and the DMD tone adjustment function and the color specification of the filter that can be switched at high speed are synchronized to project colors by region is as follows.

When visualizing intracellular responses to extracellular stimuli, DMD and filters are used to light-release stimuli of caged compounds at specific wavelengths (colors) in specific areas outside the cells. In this specific region, fluorescence is observed at a specific excitation wavelength. In addition, if the stimulus-response response is very fast, it is necessary to switch at high speed. Industrial applicability

[0020] This is a two-puck confocal microscope that can select the irradiation area of a specimen by DMD, and can be applied in a wide range of fields such as medicine and bioresearch.

Claims

The scope of the claims

[1] a light source device;

A number of micromirrors arranged in two dimensions are provided, and the selected micromirrors convert the light from the light source device into dotted illumination light to generate parallel light beams according to the selected set of micromirrors. DMD to form,

It comprises a first-puku plate having a number of pinholes and a second second-puk plate having a number of microlenses, and the parallel light beam from the DMD is converged by the microlenses of the second-puk plate and the first -Forming an image on the pinhole of the Pukou plate-Sharing the image of the light beam imaged in the pinhole plate assembly and the first-Pukou plate at the corresponding position of the field of view and reversing the fluorescence from the field of view Focusing optics,

Two-dimensional light detection means;

An output optical system comprising a color dividing optical element disposed between the first and second second plate and a lens system that forms an image of reflected light from the color dividing optical element on the light detection means; A confocal microscope, comprising: a control device that selects and controls on / off of a mirror; and a portion of the entire field of view is selected to provide illumination.

2. The confocal microscope according to claim 1, wherein the two-piece plate assembly is rotated at a high speed to form an image on the light detection means.

3. The confocal microscope according to claim 1, wherein a fluorescent specimen is placed in the field of view.