JP2009008739A - Living body observation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To make low-magnification observation and photograph return light from a single specimen by a plurality of imaging means. <P>SOLUTION: This living body observation apparatus 1 comprises a stage 2 on which the specimen A is mounted, an illumination light source 3 for emitting illumination light for irradiating the specimen mounted on the stage 2, an objective lens 4 for collecting return light from the specimen A, a beam splitter 5 for plurally branching the return light collected by the objective lens 4, a plurality of imaging lenses 16, 17 for imaging the return light branched by the beam splitter 5, respectively, and a plurality of imaging means 10, 11 for photographing observation images of the specimen imaged by the respective imaging lenses 16, 17. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a living body observation apparatus.

Conventionally, an observation apparatus for observing a sample stained with a plurality of fluorescent dyes or a sample expressing a plurality of fluorescent proteins is known (see, for example, Patent Document 1).
This observation device irradiates a sample with light of a plurality of wavelengths at the same time, collects light returning from the sample with an objective lens, and branches by wavelength by a dichroic mirror while being imaged by an imaging lens. The image is picked up by a plurality of image pickup means.

Japanese Patent Laid-Open No. 2004-177661

  However, although the observation apparatus disclosed in Patent Document 1 separates the return light from the sample collected by the objective lens between the imaging lens and the imaging means, it has a low magnification like a living body observation apparatus. When a reduction optical system is required, it is difficult to secure a large space between the imaging lens and the imaging means, and there is a disadvantage that a similar mechanism cannot be configured.

  The present invention has been made in view of the above-described circumstances, and provides a living body observation apparatus that enables observation at a low magnification and can capture return light from the same sample by a plurality of imaging means. It is intended to provide.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a stage on which a sample is mounted, an illumination light source that generates illumination light for irradiating the sample mounted on the stage, an objective lens that collects return light from the sample, and a collection by the objective lens. A beam splitter that divides the returned light into a plurality of beams, a plurality of imaging lenses that respectively image the return light branched by the beam splitter, and an observation image of the sample imaged by each imaging lens A living body observation apparatus including a plurality of imaging means is provided.

  According to the present invention, when the illumination light emitted from the illumination light source is irradiated onto the sample mounted on the stage, the return light from the sample is collected by the objective lens, and the collected return light is collected by the beam splitter. A plurality of optical paths are branched. Each branched return light is imaged by a separate imaging lens and then photographed by a plurality of imaging means. Thereby, a plurality of observation images can be acquired. In this case, since the imaging lens is provided separately, the distance required for imaging the return light by the imaging lens can be made different when acquiring observation images with different magnifications. And unlike the conventional device that branches between the imaging lens and the imaging means, it branches by the beam splitter in front of the imaging lens, so when acquiring a low magnification observation image, The interval between the imaging means can be set short.

In the above invention, the illumination light source generates illumination light including excitation light, and a filter that transmits one of reflected light, fluorescence, and light emission from the sample is provided between the beam splitter and the plurality of imaging units. It may be arranged.
By doing so, it is possible to select one of reflected light, fluorescence, and light emission from the sample with the filter and take an image with each imaging means. Then, by displaying the reflected light image obtained separately and either the fluorescence image or the luminescence image in an overlapping manner, it is possible to confirm at a glance from which part of the sample the fluorescence or luminescence is emitted. Moreover, it is possible to observe in real time without switching the imaging means.

In the above invention, the plurality of imaging means may be imaging means capable of acquiring a monochrome image and imaging means capable of acquiring a color image.
By doing in this way, a color image or a monochrome image can be acquired according to a use. That is, a color image has an advantage that an observation image can be easily seen, and a monochrome image has an advantage that luminance analysis can be facilitated. Therefore, by providing both, it can be applied to various applications.

In the invention described above, the plurality of imaging lenses may have different focal lengths.
By doing in this way, the distance between each imaging lens and each imaging means can be varied, and observation images with different magnifications can be observed simultaneously.

  According to the present invention, it is possible to perform observation at a low magnification, and it is possible to capture the return light from the same sample with a plurality of imaging means.

Hereinafter, a living body observation apparatus 1 according to an embodiment of the present invention will be described with reference to FIG.
The living body observation apparatus 1 according to this embodiment is an apparatus for observing a living body such as a small experimental animal such as a mouse as the sample A.

  As shown in FIG. 1, the living body observation apparatus 1 according to the present embodiment generates a stage 2 on which a sample A such as an experimental small animal is mounted, and illumination light that irradiates the sample A mounted on the stage 2. The illumination light source 3, the illumination light from the illumination light source 3 is irradiated onto the sample A, while the objective lens 4 that condenses the return light from the sample A, and the return light collected by the objective lens 4 is 2 A first beam splitter 5 that branches into two, two observation optical systems 6 and 7 that respectively guide return light branched by the first beam splitter 5, and light guides through the observation optical systems 6 and 7. Filters 8 and 9 that selectively transmit part of the returned light, and two imaging means 10 and 11 that capture the return light that has passed through the filters 8 and 9 are provided.

Outputs from the imaging units 10 and 11 are connected to an image processing device 12, and the image processing device 12 is connected to a monitor 13.
In the figure, reference numeral 14 denotes an objective turret that holds a plurality of objective lenses 4 in a switchable manner, and reference numeral 19 denotes observation optical systems 6 and 7, first beam splitter 15, objective lens 4, filters 8 and 9, and sample A. Is a dark box device that surrounds the stage 2 on which is mounted and blocks the incidence of external light.

The illumination light source 3 generates illumination light in a relatively wide wavelength band including an excitation wavelength that can excite the fluorescent substance contained in the sample A.
A second beam splitter 15 is disposed between the illumination light source 3 and the objective lens 4 to reflect a part of the illumination light from the illumination light source 3 and direct it toward the objective lens 4 side. A part of the return light is transmitted and directed to the first beam splitter 5.
The first and second beam splitters 5 and 15 are, for example, half mirrors.

  The two observation optical systems 6 and 7 are provided with an optical system such as a lens (not shown) and imaging lenses 16 and 17 for collecting the return light. Each of the imaging lenses 16 and 17 focuses the condensed return light on the imaging surfaces of the imaging means 10 and 11, respectively. In the figure, reference numeral 18 denotes a mirror.

  The filters 8 and 9 allow the passage of light in the same wavelength band as the illumination light among the light returning from the sample A, and the filter 8 that prohibits the passage of other light, and the same wavelength band as the illumination light A filter 9 that prohibits the passage of other light and permits the passage of other light is disposed in a separate optical path.

The imaging means 10 and 11 are CCD cameras, respectively.
Thereby, the reflected light as the return light from the sample A is photographed by one imaging means 10 to obtain a reflected light image, and the fluorescence as the return light from the sample A is obtained by the other imaging means 11. A fluorescent image is acquired by photographing.

  The image processing device 12 is configured to superimpose the reflected light image and the fluorescence image acquired by the two imaging means 10 and 11. The monitor 13 displays the reflected light image and the fluorescence image superimposed by the image processing device 12 at the same time.

The operation of the biological observation apparatus 1 according to the present embodiment configured as described above will be described below.
In order to observe the sample A such as an experimental small animal using the living body observation apparatus 1 according to the present embodiment, the sample A is administered or injected with a fluorescent agent that specifically accumulates in a tumor tissue such as cancer. Then, the sample A that has been put into a sleep state by anesthesia is fixed to the stage 2 and the illumination light source 3 is operated to emit illumination light.
A part of the illumination light emitted from the illumination light source 3 is directed to the objective lens 4 by the second beam splitter 15 formed of a half mirror, is condensed by the objective lens 4, and is applied to the sample A on the stage 2. The

Since the illumination light includes light having an excitation wavelength, the fluorescent agent contained in the sample A is excited by the irradiated light having the excitation wavelength, and fluorescence having a predetermined wavelength is generated. A part of the irradiated illumination light is reflected on the surface of the sample A.
Therefore, the fluorescence generated from the sample A and the reflected light on the surface of the sample A are collected as return light by the objective lens 4, and a part of the light passes through the second beam splitter 15 and passes through the first beam splitter 5. And is split into two optical paths by the first beam splitter 5.

  Observation optical systems 6 and 7 are arranged in each optical path, and the respective light is condensed by the imaging lenses 16 and 17 included in the observation optical systems 6 and 7. The lights collected by the imaging lenses 16 and 17 are transmitted through the filters 8 and 9, respectively, so that only predetermined wavelength components are allowed to pass therethrough and other wavelength components are prohibited from passing.

  Since the filter 9 in one optical path allows only the fluorescence to pass and prohibits the passage of light in other wavelength bands, the fluorescent image that has passed is imaged on the imaging surface of the imaging means 11 to obtain a fluorescence image. The The filter 8 in the other optical path prohibits the transmission of fluorescence and allows the passage of light in other wavelength bands, so that the reflected light that has passed through is imaged on the imaging surface of the imaging means 10, thereby reflecting the reflected light image. Is acquired.

Then, the fluorescence image and the reflected light image acquired by each of the imaging units 10 and 11 are superimposed on the image processing device 12 and then displayed on the monitor 13 at the same time.
Thereby, the observer can observe the superimposed image of the fluorescent image and the reflected light image on the monitor 13 at the same time, and while looking at the appearance of the sample A displayed by the reflected light image, The position and size of the displayed tumor or the like can be easily confirmed. In particular, there is an advantage that both images can be observed simultaneously in real time by superimposing and displaying the fluorescent image and the reflected light image.

  In this case, according to the living body observation apparatus 1 according to the present embodiment, the return light from the sample A is branched at the front stage of the imaging lenses 16 and 17, so that the reduction optical system with a low magnification is used as the observation optical systems 6 and 7. Even when the distance between the imaging lenses 16 and 17 and the imaging means 10 and 11 cannot be increased, the fluorescence image and the reflected light image can be observed separately at the same time.

  In the present embodiment, the return light is branched by the first beam splitter 5 made of a half mirror, but instead, a dichroic mirror may be adopted. For example, when the wavelength of the excitation light is 460 to 490 nm and the wavelength of the generated fluorescence is 510 nm or more, by adopting a dichroic mirror that branches at 505 nm as a boundary, the return light is branched without waste, and bright fluorescence Images and reflected light images can be acquired.

  Further, in the present embodiment, as shown in FIG. 2, a beam splitter 5 made of a half mirror may be provided so as to be inserted into and removed from the optical path of the return light condensed by the objective lens 4. By doing in this way, all the return lights can be imaged by one imaging means 10, and a bright fluorescent image can be acquired.

  In the present embodiment, the focal lengths of the imaging lenses 16 and 17 provided in the two observation optical systems 6 and 7 may be different. By doing in this way, the magnification of the fluorescence image and reflected light image obtained by the two imaging means 10 and 11 can be varied. In this case, the image processing device 12 may perform image processing so that two images are simultaneously displayed in separate windows on the monitor 13 without overlapping the two images.

  The imaging lenses 16 and 17 may be provided so as to be movable along the optical axis direction. In this way, focusing can be performed by adjusting the imaging position by the imaging lenses 16 and 17 in the optical axis direction.

  Moreover, you may decide to have the turret (not shown) which hold | maintains the several imaging lenses 16 and 17 so that switching is possible. Thereby, the observation magnification can be easily changed by rotating the turret and replacing the imaging lenses 16 and 17.

  Further, as the imaging means 10 and 11, one that acquires a color image and one that acquires a monochrome image may be adopted. According to a color image, it is easy to visually recognize an observation image, and according to a monochrome image, there is an advantage that luminance analysis can be easily performed.

  In the embodiments described so far, the case where a fluorescent image and a reflected image are acquired has been described. However, a luminescent image and a reflected image may be separately observed simultaneously using a luminescent gene such as luciferase. Moreover, you may observe a fluorescence image and a light emission image separately separately. In this case, it is preferable to confirm the localization with the fluorescence image and to confirm the quantitativeness with the emission image.

It is a whole lineblock diagram showing a living body observation device concerning one embodiment of the present invention. It is a whole block diagram which shows the modification of the biological observation apparatus of FIG.

Explanation of symbols

A Sample 1 Living body observation apparatus 2 Stage 3 Illumination light source 4 Objective lens 5 First beam splitter (beam splitter)
8, 9 Filter 10, 11 Imaging means 16, 17 Imaging lens

Claims (4)

A stage on which the sample is mounted;
An illumination light source that generates illumination light for irradiating a sample mounted on the stage;
An objective lens that collects the return light from the sample;
A beam splitter for branching the return light collected by the objective lens into a plurality of parts,
A plurality of imaging lenses that respectively image the return light branched by the beam splitter;
A biological observation apparatus comprising: a plurality of imaging units that capture observation images of a sample imaged by each imaging lens. The illumination light source generates illumination light including excitation light;
The living body observation apparatus according to claim 1, wherein a filter that transmits one of reflected light, fluorescence, and luminescence of the sample is disposed between the beam splitter and the plurality of imaging units.   The living body observation apparatus according to claim 1, wherein the plurality of imaging units are an imaging unit capable of acquiring a monochrome image and an imaging unit capable of acquiring a color image.   The living body observation apparatus according to claim 1, wherein the plurality of imaging lenses have different focal lengths.

Images