JP5257605B2 - Confocal microscope - Google Patents

Description

  The present invention relates to a confocal microscope apparatus using a rotating pinhole array disk, and more particularly to a confocal microscope apparatus capable of acquiring both a confocal image and a bright field image of a cell.

  Conventionally, analysis of target movement and increase / decrease in quantity is performed by fusing fluorescent protein with a target in the target cell and acquiring a fluorescence image of the cell cross section with a confocal microscope. .

  However, only the fluorescence image reveals the target movement, but it is unclear where this is in the cell. Therefore, in order to observe the morphology of the whole cell, it is necessary to acquire a bright field image (generally, there are many phase difference images). That is, by obtaining both the fluorescence image and the bright field image, it is possible to confirm in which part of the cell the target is located.

  FIG. 4 is a configuration explanatory view showing a conventional confocal microscope apparatus described in Patent Document 1 described later.

A sample 2 in one of the arrayed wells (holes) in the well plate 1 is irradiated with bright field illumination light 3 emitted from a halogen lamp (not shown). The irradiated sample 2 forms a bright field image by the objective lens 6 of the microscope 5. This image is formed on the camera 11 by the variable power lens 10 disposed on the lens holder 9. Since the magnification of the zoom lens 10 is a reduction system, the camera 11 becomes a bright field image obtained by imaging a wide range of the sample.

  Here, in order to separate from the optical path of the confocal image described later, the optical path of the bright-field image is reflected using the dichroic mirror 8 in the connecting member 7 disposed in the optical path. As the wavelength characteristics of the dichroic mirror 8, it is desirable to reflect the long wavelength light and transmit the short wavelength light. For example, if a dichroic mirror that reflects light having a wavelength of 680 nm or more is used, the fluorescence signal from the sample 2 is transmitted through the dichroic mirror 8 without being lost, and the confocal optical system configured by the confocal scanner 13 and the spectroscopic optical unit 20 is used. Reach the system.

  On the other hand, in the optical path for obtaining a confocal image, the confocal scanner 13 is connected to the microscope 5 via the connecting member 7, and the parallel excitation light beam 12 for illumination (a one-dot chain line) is a microlens array disk (referred to as ML disk) 14. Is transmitted through a first dichroic mirror (DM) 15 formed of a flat mirror having spectral characteristics, and passes through individual pinholes of the Nipkow disk 16 to pass through the dichroic mirror 8 of the connecting member 7. To Penetrate. Further, the sample is condensed on each sample 2 of the sample 2 placed on the well plate 1 by the objective lens 6 of the microscope 5 to excite the fluorescent reagent. The ML disk 14 and the Niipou disk 16 rotate about the support shaft 17.

  In FIG. 4, reference numeral 20 denotes a spectroscopic optical unit connected to the confocal image extraction port of the confocal scanner 13. The spectroscopic optical unit 20 includes a dichroic mirror 22 including a flat mirror as return light separating means and bandpass filters 23a and 23b having spectral characteristics as stray light removing means.

  Each sample of the sample 2 is added with, for example, fluorescent reagents CY3 and CY5, and the fluorescent signal 18 emitted from each fluorescent reagent passes through the objective lens 6 again and is collected on individual pinholes of the Niipou disc 16. The fluorescent signals 18 that have passed through the individual pinholes are reflected by the dichroic mirror 15, and confocal optical images of the selected wavelengths are respectively transmitted to the cameras 24a and 24b via the relay lens 19, the lens 21a, and the bandpass filters 23a and 23b. Is imaged.

  In the above-described configuration, the plane on which the pinholes of the Niipou disk 4 are arranged, the plane to be observed on the sample 2, and the light receiving surfaces of the cameras 24a and 24b are arranged in an optically conjugate relationship with each other. An optical cross-sectional image of the sample 2, that is, a confocal image is formed on 24a and 24b. Accordingly, since a confocal image of the sample 2 can be simultaneously formed on the light receiving surfaces of the cameras 24a and 24b, the sample 2 in which a large number of samples to be inspected are arranged on a matrix is relatively relative to the microscope and the confocal scanner. By moving to the position, it is possible to capture a confocal image obtained by performing arbitrary wavelength selective separation of all the samples at high speed.

Based on the bright field image obtained by the camera 11, a place where samples (cells) are present in an appropriate quantity is specified, and the well plate 1 is configured so that these samples can be imaged by the cameras 24 a and 24 b of the confocal optical system. Can be finely adjusted.

In the prior art, two cameras, 24a and 24b, are used to simultaneously acquire two colors of fluorescent images. However, when only one color is acquired, only the camera 24b is configured.

  The following patent documents are known as prior art of such a confocal microscope apparatus.

JP 2008-059872A Japanese Utility Model Publication No. 5-96816 JP 2008-249965 A

However, in Patent Document 1, as shown in FIG. 4 as the prior art, since separate cameras are used to acquire the fluorescence image and the bright field image of the cell, the microscope apparatus becomes large in size and increases in cost. There is a problem of becoming.

  Further, in Patent Document 2, since the second branch optical system is detachable, high speed cannot be realized.

  Further, since the optical element of the second branch optical system is installed in a finite system, not in an infinite optical system of a microscope, there is a defect that a ghost image is generated.

  Further, in Patent Document 3, there is a problem that high-speed performance cannot be realized because the bright-field optical path is a switching method, and a mechanism is complicated and the cost is increased because a drive system is required.

  The present invention was made to solve the above-described problems of the prior art, and devised an optical element such as a dichroic mirror used in the configuration of the optical system and the optical system, with a simple structure, with the same camera, The object is to realize a confocal microscope apparatus capable of acquiring both a fluorescence image and a bright field image of a cell.

In order to achieve such a problem, the invention according to claim 1 of the present invention is:
In a confocal microscope apparatus that acquires a fluorescence image and a bright field image based on light obtained through an objective lens from a sample that is scanned by excitation light that has passed through a rotating pinhole array disk and irradiated with bright field illumination,
A first infinity optical system for injecting light from a sample;
A first separation unit installed in the first infinity optical system and separating light from the sample into fluorescent light and bright field light by spectral characteristics that transmit or reflect according to wavelength ;
A second infinity optical system that emits bright field light emitted from the first separation means via the first infinity optical system and fluorescent light that passes through the pinhole array disk; ,
A multiplexing means installed in the second infinity optical system, for combining the fluorescent light and the bright field light by the spectral characteristics ;
Wavelength selection means for emitting either fluorescent light or bright field light emitted from the multiplexing means;
And a first camera that acquires an image output from the wavelength selection unit via the second infinity optical system.

The invention according to claim 2
The confocal microscope apparatus according to claim 1,
The first infinity optical system is:
An objective lens that converts the light from the sample into parallel light and emits it to the first separating means;
A first imaging lens that converges the fluorescent light emitted from the first separation means and guides it to the pinhole array disk;
And a second imaging lens that converges the bright field light emitted from the first separation unit and guides the bright field light to the second infinity optical system.

The invention described in claim 3
The confocal microscope apparatus according to claim 1 or 2,
The second infinity optical system is
A first relay lens that guides fluorescence light emitted from the pinhole array disk to parallel light as parallel light;
A second relay lens that guides the bright field light emitted from the second imaging lens to parallel light as parallel light;
And a third relay lens for converging the light emitted from the first wavelength selection means and entering the first camera.

The invention according to claim 4
The confocal microscope apparatus according to claim 3,
Second separation means that transmits the excitation light, reflects the fluorescent signal that has passed through the pinhole of the pinhole array disk, and guides it to the first relay lens;
And a reflection mirror that reflects the bright field light emitted from the first separation means and enters the second relay lens.

The invention according to claim 5
The confocal microscope apparatus according to claim 3 or 4,
Third separation means for wavelength-separating a part of the fluorescence signal emitted from the multiplexing means;
Second wavelength selection means for transmitting the fluorescence signal emitted from the third separation means;
A fourth relay lens for converging the light emitted from the second wavelength selection means;
And a second camera that receives light emitted from the fourth relay lens.

The invention described in claim 6
The confocal microscope apparatus according to any one of claims 1 to 5,
A dichroic mirror is used as the separating unit and the combining unit.

The invention described in claim 7
The confocal microscope apparatus according to any one of claims 1 to 5,
A dichroic mirror having the same spectral characteristics is used as the first separating unit and the combining unit.

The invention described in claim 8
The confocal microscope apparatus according to claim 7,
The spectral characteristics of the dichroic mirror are characterized by reflecting a long wavelength and transmitting a short wavelength.

The invention according to claim 9
The confocal microscope apparatus according to claim 7,
The spectral characteristics of the dichroic mirror are characterized by reflecting a short wavelength and transmitting a long wavelength.

The invention according to claim 10 is:
The confocal microscope apparatus according to any one of claims 1 to 9,
A band pass filter is used as the wavelength selection means.

The present invention has the following effects.
That is, the bright field image and the fluorescence image are divided by the wavelength band, and both the bright field image and the fluorescence image can be acquired with one camera by devising the optical path and the optical element of the two wavelengths.
As a result, the configuration of the confocal microscope apparatus can be simplified, and a highly functional apparatus can be realized at a low cost.

It is composition explanatory drawing which shows one Example of the confocal microscope apparatus which concerns on embodiment of this invention. It is composition explanatory drawing which shows the 2nd Example of the confocal microscope apparatus based on embodiment of this invention. It is composition explanatory drawing which shows the modification of the confocal microscope apparatus based on embodiment of this invention. It is composition explanatory drawing which shows the conventional confocal microscope apparatus.

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

FIG. 1 is a configuration explanatory view showing the configuration of an optical system that is the core of one example of a confocal microscope apparatus according to an embodiment of the present invention. The same elements as those in FIG.

  In FIG. 1, a dichroic mirror 80a constitutes a first separating unit, reflects long-wavelength bright-field light included in parallel light incident from the sample 2 via the objective lens 6, and emits short-wavelength fluorescence. By transmitting the light, the wavelength is separated into a bright-field optical path and a fluorescent optical path. For example, when a wavelength of 400 nm to 700 nm is set as a fluorescent image region and a wavelength of 700 nm to 750 nm is set as a bright field image region, the dichroic mirror 80a reflects light having a wavelength of 700 nm or more and transmits light having a wavelength of 700 nm or less. Has characteristics.

The first imaging lens 26 a converges the parallel light beam composed of the fluorescent light 18 transmitted through the dichroic mirror 80 a and guides it to the pinhole array disk (Nipou disk) 16 of the confocal scanner 13.

The dichroic mirror 15 constitutes a second separation means, transmits the excitation light beam 12 from a laser light source (not shown), and reflects the fluorescent signal 18 that has passed through the pinholes of the pinhole array disk 16 to reflect the first. 1 to the relay lens 29a.

The first relay lens 29a converts the fluorescent light emitted from the pinhole array disk 16 and reflected by the dichroic mirror 15 into a parallel light flux.

The second imaging lens 26b converges the bright field light reflected by the dichroic mirror 80a.

The reflection mirror 27 reflects the bright field light emitted from the second imaging lens 26b and guides it to the second relay lens 29b.

The second relay lens 29b converts the bright field light reflected by the reflection mirror 27 into parallel light.

The dichroic mirror 80b constitutes a multiplexing unit, and combines the bright field light emitted from the second relay lens 29b and the fluorescent light emitted from the first relay lens 29a. Here, by making the 80b have the same spectral characteristics as the dichroic mirror 80a, it is possible to share parts.

The bandpass filter 30a constitutes a first wavelength selection unit, and transmits either the fluorescent signal or the bright field signal light emitted from the dichroic mirror 80b and blocks the other. Here, the bandpass filter 30a is attached to a rotating filter wheel (not shown), and when acquiring a bright field image, has a characteristic of transmitting only a band of 700 nm to 750 nm, and when acquiring a fluorescence image, It has the characteristic of transmitting a wavelength band that matches the fluorescence spectrum.

The third relay lens 29c converges the parallel light emitted from the bandpass filter 30a.

The first camera 31a receives the light converged by the third relay lens 29c.

In the above description, the dichroic mirrors 80a and 80b have exactly the same spectral characteristics and reflect long wavelengths and transmit short wavelengths. However, the purpose is to minimize the types of components, so they do not necessarily have exactly the same characteristics. May be.

The first dichroic mirror 80a is installed in a first infinity optical system including the objective lens 6 and the imaging lenses 26a and 26b.
The second dichroic mirror 80b and the bandpass filter 30a are installed in a second infinity optical system composed of the relay lens pairs 29a and 29c and 29b and 29c.

Here, the infinity optical system is also called a telecentric optical system, and refers to an optical system in which the entrance pupil or the exit pupil is on the focal plane of the optical system. That is, a telecentric optical system is configured between the objective lens 6 and the imaging lens 26a and between the objective lens 6 and the imaging lens 26b. Similarly, a telecentric optical system is configured between the relay lenses 29a and 29c and between the relay lenses 29b and 29c.

  In addition, it is assumed that a method for installing, fixing, and replacing individual optical elements such as a lens, a mirror, and a filter in the optical system is performed by a general method, and description thereof is omitted here.

The operations for acquiring the bright field image and the fluorescence image in the apparatus of FIG. 1 will be described below.

  First, acquisition of a bright field image will be described.

When a sample 2 such as a cell disposed on the well plate 1 is irradiated with a bright field illumination 3 emitted from a bright field light source (not shown), bright field signal light 4 is obtained. This signal light is collected by the objective lens 6, reflected by the dichroic mirror 80a, and guided to the bright field optical path.

  The bright field signal light 4 reflected by the dichroic mirror 80a is converged by the second imaging lens 26b, reflected by the reflecting mirror 27, and then forms an image on the imaging surface 28b. Here, the reason why the traveling direction of the bright field signal 4 is changed using the reflection mirror 27 is to mix the bright field signal 4 with the optical path of the fluorescence signal.

The bright field image on the image plane 28b becomes parallel light by the relay lens 29b, is reflected by the dichroic mirror 80b, passes through the bandpass filter 30a, is converged by the relay lens 29c, and enters the first camera 31a. When acquiring a bright field image, the band pass filter 30a is selected to have a characteristic of transmitting only the band of 700 nm to 750 nm. Here, since the optical elements 80b and 30a are installed between the telecentric relay lenses 29b and 29c, a ghost image is not generated by multiple reflection of light generated on the surface of the optical element. This is because parallel light generated by multiple reflection of light between telecentric relay lenses converges to one point via the relay lens.

Next, acquisition of a fluorescence image will be described.

As shown in FIG. 1, a sample 2 such as a cell placed on a well plate 1 is irradiated with an excitation light beam 12 emitted from a laser light source (not shown) in which a plurality of wavelengths are mixed. An excitation light beam 12 emitted from a laser light source (not shown) is collected by a microlens array disk 14, passes through a dichroic mirror 15, passes through a pinhole of a Nipkow disk 16, becomes parallel light by an imaging lens 26 a, and becomes dichroic. The light passes through the mirror 80a, is converged by the objective lens 6, and is irradiated onto the sample 2.

The fluorescent signal 18 generated from the irradiated sample 2 is condensed by the objective lens 6, converted into parallel light, transmitted through the dichroic mirror 80a, converged by the imaging lens 26a, and a fluorescent image is formed on the imaging surface 28a. . Since the pinhole array disk 16 with microlenses is arranged so that the pinhole surface coincides with the image plane 28a, the fluorescent image becomes a confocal image. The confocal image is reflected by the dichroic mirror 15 and formed on the camera 31a by the telecentric relay lens systems 29a and 29c. At this time, the dichroic mirror 80b transmits the fluorescence wavelength as described above, and the band-pass filter 30a has its characteristics switched by the filter wheel, and transmits only the fluorescence wavelength band corresponding to the wavelength of the excitation light beam.

  When acquiring an image as described above, only an excitation light beam is input when acquiring a fluorescent image, and only bright field illumination is input when acquiring a bright field image. After acquiring two types of images, a bright-field image and a fluorescence image, in a time-division manner, they are superimposed using an image processing device or the like.

According to the confocal microscope apparatus as described above, the bright field image and the fluorescence image are divided by the wavelength band, and the light path image and the fluorescence image are obtained with one camera by devising the optical path and the optical element of the two wavelengths. Can get both. As a result, the configuration of the confocal microscope apparatus can be simplified, and a highly functional apparatus can be realized at a low cost.

Further, since an infinite optical system is used, there is an advantage that the expandability of the optical element inserted between the lenses can be increased.

  In addition, when acquiring multi-color fluorescence, you may acquire a multiple types of fluorescence image by a time division by switching an excitation wavelength sequentially and switching the band pass filter 30a corresponding to it. In this case, usually, a plurality of images are acquired for each sample.

  FIG. 2 shows a second example of the confocal microscope apparatus according to the embodiment of the present invention, in which a dichroic mirror is installed between the telecentric relay lenses of FIG. 1 and two-color fluorescence images are simultaneously acquired. It is a configuration explanatory view showing what can be. The same elements as those in FIG.

In FIG. 2, the dichroic mirror 32 constitutes a third separation means for reflecting a part of the fluorescent signal emitted from the dichroic mirror 80b to separate the wavelength. The bandpass filter 30b constitutes a second wavelength selection unit that transmits the fluorescent signal reflected by the dichroic mirror 32. The relay lens 29d constitutes a fourth relay lens that converges the fluorescent light emitted from the bandpass filter 30b. The camera 31b constitutes a second camera that receives light emitted from the relay lens 29d.

The operation of the apparatus of FIG. 2 will be described below.

Due to the spectral characteristics of the dichroic mirror 32, the fluorescence signal emitted from the dichroic mirror 80b is transmitted with long wavelength fluorescence and reflected with short wavelength fluorescence.

As an example, when the dichroic mirror 32 transmits mRFP fluorescence and reflects GFP fluorescence, two-wavelength lasers of 473 nm and 561 nm are excited simultaneously as excitation light beams, and the bandpass filters 30a and 30b are respectively mRFP and GFP. Transmits fluorescence. As a result, the fluorescence image of mRFP is acquired from the camera 31a, and the fluorescence image of GFP is acquired from the camera 31b, so that two-color fluorescence images can be acquired simultaneously.

According to the confocal microscope apparatus of FIG. 2, simultaneity between two color fluorescence images is ensured even when the sample moves fast.

If the device of FIG. 2 has a plurality of waveband filters mounted on the filter wheel as the bandpass filters 30a and 30b, a larger number of fluorescent images can be obtained.

  FIG. 3 is a structural explanatory diagram showing a modification of the confocal microscope apparatus according to the embodiment of the present invention, in which the spectral characteristics of the dichroic mirror that separates the bright-field optical path and the fluorescent optical path are reversed from those in FIG. . The same elements as those in FIG.

In the apparatus of FIG. 3, the spectral characteristics of the dichroic mirrors 180a and 180b are configured to reflect short wavelength light and transmit long wavelength light. As an example of the spectral characteristic, there is one that reflects light of 400 nm to 700 nm and transmits light of 700 nm to 750 nm.

The operation of the apparatus of FIG. 3 will be described below with a focus on the differences from the operation of FIG. Other operations are the same as those in FIG.

The excitation light beam 12 exiting the imaging lens 26 a is reflected by the dichroic mirror 180 a and enters the objective lens 6. The fluorescence signal 18 emitted from the objective lens 6 is reflected by the dichroic mirror 180a and enters the imaging lens 26a. The fluorescent light converged by the imaging lens 26a is reflected by the dichroic mirror 15 and then enters the relay lens 29a. The fluorescent signal 18 emitted from the relay lens 29a is reflected by the dichroic mirror 180b and enters the bandpass filter 30a. To do.

The bright-field signal light 4 emitted from the objective lens 6 passes through the dichroic mirror 180a, forms an image on the coupling surface 128b by the imaging lens 26b, is reflected by the reflection mirror 27, and is guided to the relay lens 29b. . The bright field signal light 4 emitted from the relay lens 29b passes through the dichroic mirror 180b and enters the bandpass filter 30a.

The confocal microscope apparatus of FIG. 3 produces the same effect as that of FIG.

As in the case of FIG. 1, a plurality of types of fluorescent images may be acquired in a time division manner.

  Further, similarly to FIG. 2, elements may be added to obtain two colors of fluorescent images at the same time.

  The present invention is not limited to the above-described embodiments and modifications, and includes many changes and modifications without departing from the essence thereof.

2 Sample 3 Bright field illumination 4 Bright field signal light 6 Objective lens 12 Excitation light 15 Second separation means 16 Pinhole array disk 18 Fluorescent signal 26a First imaging lens 26b Second imaging lens 27 Reflecting mirror 29a First 1 relay lens 29b 2nd relay lens 29c 3rd relay lens 29d 4th relay lens 30a 1st wavelength selection means 30b 2nd wavelength selection means 31a 1st camera 31b 2nd camera 32 3rd Separating means 80a First separating means 80b Multiplexing means

Claims (10)

In a confocal microscope apparatus that acquires a fluorescence image and a bright-field image based on light obtained through an objective lens from a sample that is scanned with excitation light that has passed through a rotating pinhole array disk and irradiated with bright-field illumination,
A first infinity optical system for injecting light from a sample;
A first separation unit installed in the first infinity optical system and separating light from the sample into fluorescent light and bright field light by spectral characteristics that transmit or reflect according to wavelength ;
A second infinity optical system that emits bright field light emitted from the first separation means via the first infinity optical system and fluorescent light that passes through the pinhole array disk; ,
A multiplexing means installed in the second infinity optical system, for combining the fluorescent light and the bright field light by the spectral characteristics ;
Wavelength selection means for emitting either fluorescent light or bright field light emitted from the multiplexing means;
A confocal microscope apparatus comprising: a first camera that acquires an image output from the wavelength selection unit via the second infinity optical system. The first infinity optical system is:
An objective lens that converts the light from the sample into parallel light and emits it to the first separating means;
A first imaging lens that converges the fluorescent light emitted from the first separation means and guides it to the pinhole array disk;
The confocal microscope apparatus according to claim 1, further comprising: a second imaging lens that converges bright field light emitted from the first separation unit and guides the bright field light to the second infinity optical system. . The second infinity optical system is
A first relay lens that guides fluorescence light emitted from the pinhole array disk to parallel light as parallel light;
A second relay lens that guides the bright field light emitted from the second imaging lens to parallel light as parallel light;
The confocal microscope apparatus according to claim 1, further comprising a third relay lens that converges the light emitted from the first wavelength selection unit and enters the first camera. Second separation means that transmits the excitation light, reflects the fluorescent signal that has passed through the pinhole of the pinhole array disk, and guides it to the first relay lens;
4. The confocal microscope apparatus according to claim 3, further comprising a reflection mirror that reflects the bright field light emitted from the first separation unit and enters the second relay lens. Third separation means for wavelength-separating a part of the fluorescence signal emitted from the multiplexing means;
Second wavelength selection means for transmitting the fluorescence signal emitted from the third separation means;
A fourth relay lens for converging the light emitted from the second wavelength selection means;
The confocal microscope apparatus according to claim 3, further comprising a second camera that receives light emitted from the fourth relay lens. 6. The confocal microscope apparatus according to claim 1, wherein a dichroic mirror is used as the separating unit and the combining unit. 6. The confocal microscope apparatus according to claim 1, wherein a dichroic mirror having the same spectral characteristics is used as the first separating unit and the combining unit. 8. The confocal microscope apparatus according to claim 7, wherein the spectral characteristics of the dichroic mirror reflect a long wavelength and transmit a short wavelength. 8. The confocal microscope apparatus according to claim 7, wherein the spectral characteristic of the dichroic mirror reflects a short wavelength and transmits a long wavelength. The confocal microscope apparatus according to claim 1, wherein a band pass filter is used as the wavelength selection unit.

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