HK1130906A

HK1130906A - Observing tool and observing method using same

Info

Publication number: HK1130906A
Application number: HK09109446.2A
Authority: HK
Inventors: 金崎士朗; 林真市
Original assignee: 株式会社Eci
Priority date: 2003-03-27
Filing date: 2009-10-13
Publication date: 2010-01-08

Description

Observation tool and observation method using the same

Technical Field

The present invention relates to a technique for observing an object, and more particularly to a technique for observing a transparent fine object such as a cell.

Background

Conventionally, a transmission observation microscope is used for observing a minute object such as a cell. In order to observe the internal structure and morphology of a living cell without staining with high contrast, it is necessary to use a phase difference microscope, a non-output phase difference microscope, a differential interference microscope, a polarized light microscope, or the like, which is provided with a special condenser having a phase difference ring, a differential interference prism, or the like, and which has a special lens.

Specifically, as a method for observing transparent fine objects such as cells in a culture medium under a microscope, special observation methods such as a phase difference method, an oblique illumination method, and a differential interference method are widely used (see, for example, Japanese patent laid-open No. 7-225341). The structure of an optical system for special observation is shown in fig. 16, and a general structure for transmission clear field observation is composed of: a light source 713 for generating illumination light; an aiming lens 741 for aligning the directions of the illumination lights emitted from the light sources 713; a mirror 742 for deflecting the traveling direction of the illumination light whose traveling direction matches in the vertical direction; a window lens 743 for condensing the illumination light; a condenser lens 744 for irradiating the condensed illumination light to the specimen 708 containing the observation object; an objective lens 706 for enlarging the projection sample 708; an imaging lens 746 for imaging an image of the sample 708 onto an imaging plane 745; and a stage 717 for adjusting the observation position of the specimen 708. In this configuration, an illumination modulation element 747 is added at the position of the entrance hole of the condenser lens 744, and an imaging modulation element 748 is added at the position of the exit hole of the objective lens 706. The phase difference method is used for the illumination modulation element 747 using a ring slit and the imaging modulation element 748 using a phase plate, the oblique illumination method is used for the illumination modulation element using an eccentric aperture, and the differential interference method is used for the illumination modulation element 747 and the imaging illumination element 748 using a polarizing plate and a differential interference prism. By using a special observation method, even a transparent object can be observed with a contrast of light and shade if the refractive index is different from that of the surrounding medium. Such a special observation method is particularly effective in observing a living tissue which is originally transparent and difficult to see in its original state with a good contrast.

However, in these special observation methods, it is necessary to arrange a special modulation element at the aperture position of the condenser lens 744 or the objective lens 706, and these special modulation elements are very expensive, and there are many inconveniences in use, for example, a problem that the modulation element needs to be changed when the magnification of the objective lens 706 is changed, and the like.

Disclosure of Invention

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a technique capable of easily performing observation without using a special modulation element required for a special observation method when observing a transparent fine object.

The observation tool of the present invention is an observation tool having an observation target storage unit having a reflection surface. The observation tool is used for storing an observation target used in an observation method for observing an observation target by illuminating the observation target with epi-illumination light through an optical system having an objective lens, and is provided with a reflection surface for reflecting the epi-illumination light at the time of observation.

The reflecting surface may be provided on a surface facing the objective lens in observation, or may be provided on a surface opposite to the surface facing the objective lens.

In addition, a flow path through which the observation target passes may be formed. The storage unit for storing the observation target may include a port for injecting the observation target and a port for discharging the observation target.

Further, an observation method of the present invention is characterized in that: the method is a method for observing an observation target by illuminating with epi-illumination light through an optical system having an objective lens, wherein a reflection surface for reflecting the epi-illumination light at the time of observation is provided in an observation tool for storing the observation target, the observation target is stored in the observation tool, and the observation target is observed.

The observation target may be a fine transparent body.

The observation instrument may have a container capable of holding a liquid, and the container may store the liquid containing the observation target at the same time. Here, the observation target may be a cell, and the liquid may be a culture solution.

The observation target may be stored in the observation tool such that a distance between the observation target and the reflection surface is equal to or less than half a focal depth of the optical system.

Specifically, the observation target may be stored in the observation tool so that a distance d between the observation target and the reflecting surface satisfies the following formula (1).

d≦W/(2NA²)…(1)

(wherein d represents the distance between the observation target and the reflecting surface, W represents the wavelength of the light used, and NA represents the numerical aperture of the optical system.)

Further, the observation target may be housed in the observation tool so that a numerical aperture of the illumination light to face the observation target is smaller than a numerical aperture of the objective lens.

Specifically, the observation target may be housed in the observation tool so that a distance d between the observation target and the reflecting surface satisfies the following formula (2).

d>F/(4tan(sin^-1NA))…(2)

(wherein d represents a distance between an observation target and a reflecting surface, F represents a field diameter of an optical system, and NA represents a numerical aperture of the optical system.)

According to the present invention, transparent fine objects such as cells can be observed with good contrast without using a special optical unit.

That is, the cell observation instrument of the present invention can observe cells, for example, blood cells such as neutrophils, eosinophils, basophils, monocytes, macrophages, and lymphocytes, or other animal cells, or plant protoplasts, or other particle components contained therein, by using a normal optical microscope or a monitor using a normal lens and a CCD camera, and thus can observe cells without using a special device such as a phase-contrast microscope according to the related art.

Drawings

Fig. 1(a) to (C) are sectional views of the observation tool according to the first embodiment.

Fig. 2(a) and (B) are cross-sectional views of an observation tool having a structure 40 including a reflection surface.

Fig. 3(a1) to (C) are sectional views of observation tools having covers for covering the storage sections.

Fig. 4 is a cross-sectional view showing another example of the use of the observation tool according to the first embodiment, (a) is a cross-sectional view when the structure 1 is mounted on a cover glass, (B) is a schematic cross-sectional view when a hole through which a liquid can pass is provided. (C) Is an oblique view of the observation tool of (B).

Fig. 5 is a top view of an example of the observation tool having a scale mark on a storage portion.

Fig. 6(a) is a cross-sectional view of an observation tool formed in a tubular shape as a whole. (B) Is a cross-sectional view in the direction of the broken line AB of (A).

Fig. 7 is a diagram showing the structure of the observation device of the second embodiment.

Fig. 8 is a diagram showing the structure of an optical system for observation according to the present invention.

FIG. 9 is a graph showing a culture dish and cultured cells.

Fig. 10 is a diagram showing a contrast acquisition method of an object of the present invention.

Fig. 11(a) is a graph showing a calculation model of an imaging simulation in the second embodiment. Fig. 11(b) and (c) are graphs showing calculation results of imaging simulation.

Fig. 12(a) is a diagram showing an imaging simulation calculation model in the second embodiment. FIGS. 12(b) to (f) are graphs showing the calculation results of imaging simulation at distances of 1.0. mu.m, 1.2. mu.m, 1.4. mu.m, 1.6. mu.m, and 1.8. mu.m between the cell 302 and the mirror coating 307.

Fig. 13 is a graph showing a decrease in the numerical aperture of illumination light by the reflection surface.

Fig. 14(a) is a graph showing a calculation model of a simulation example of an object image in which the numerical aperture of the illumination light is smaller than the numerical aperture of the objective lens, and fig. 14(b) to (f) are graphs showing calculation results of imaging simulation in which the numerical aperture of the illumination light is 100%, 80%, 60%, 40%, and 20% of the numerical aperture of the objective lens.

Fig. 15 is a diagram showing a configuration of a minute flow path observation device according to a third embodiment.

Fig. 16 is a diagram showing the structure of an optical system according to a conventional special observation method.

Fig. 17 is a diagram showing a method of acquiring the contrast of an object in a conventional transmission observation microscope.

Fig. 18 is a photograph taken when eosinophils were observed using the observation instrument shown in fig. 3(a 2).

Detailed Description

An embodiment of the present invention will be described below with reference to the drawings. First, an observation tool to which the first embodiment of the present invention is applied will be described.

The observation tool of the present invention is a tool for observing or detecting cells or the like by reflected light using a microscope, and has a portion for storing cells or the like, that is, an observation target storage portion. The observation tool has at least a surface that reflects light in a wavelength region to be observed by the observation tool, that is, a surface on which a reflection surface is formed.

Fig. 1 to 6 show an example of the structure of the observation tool according to the present invention. In the figure, arrow X indicates observation of the observation target in the X direction by the objective lens.

In the observation tool shown in fig. 1(a), 1 is a structure made of glass, synthetic resin, metal, silicon, or the like, and 2 is a tank for storing an observation target such as a cell. A reflecting mirror that reflects light in the wavelength region of observation is provided on the bottom surface 3 of the storage section 2. Examples of the reflecting mirror constituting the reflecting surface include metal plating such as silver plating or chrome plating of glass, synthetic resin, metal, or the like, or a reflecting mirror surface formed by bonding metal foil. Further, for example, there are: the structure 1 is made of a material such as a metal or a silicon wafer which can be processed into a mirror surface, and when a groove is formed, a mirror is directly formed on the bottom surface, and after the groove is formed, the bottom surface of the groove is subjected to mirror processing or the like. Here, if at least the bottom surface 3 is a mirror, all the surfaces of the structure may be mirrors.

When the structure 1 shown in FIG. 1(A) is formed of a silicon wafer, the silicon wafer can be processed by a conventional method such as mechanical polishing or chemical etching, and can be produced by forming a groove for storing cells. For example, in the case of FIG. 1(A), the mirror 3 is formed by making the bottom surface of the silicon wafer mirror-like. Further, when the mirror surface state cannot be sufficiently expressed by the groove forming step, the bottom surface of the groove may be further processed to form a mirror surface by a step as necessary.

For example, in order to observe the granular structure of cells in detail, the depth of the storage part is preferably a depth at which the cells are arranged approximately one layer. For example, the depth of the storage part is 1 to 100 μm, and the diameter is 1 to 5mm in the case of a circular shape, but the invention is not limited thereto, and an appropriate size can be selected as required.

The material of the structure 1 itself may be a mirror surface (reflection surface) processed, or plated to form a reflection surface. It is possible to use: inorganic compounds such as glass and quartz; plastics such as polystyrene, synthetic resin, polypropylene, polyethylene, vinyl chloride, polyphenylene ether, polyphenylene sulfide, etc.; metals or alloys such as stainless steel, aluminum, bronze, etc.; non-metallic inorganic materials such as ceramics.

The observation tool shown in fig. 1(B) is configured by, for example, a structure 1 made of glass, plastic, or the like, and is provided with a storage section 2, and a reflective layer made of a material having a smooth surface and a high reflectance is formed on a bottom section 4 of the storage section 2. For example, a foil or film of bonded silicon wafers, silver plating, and the like.

The observation tool shown in fig. 1(C) is characterized in that: a reflection surface is provided on a surface 41 opposite to the surface facing the objective lens, and the structure 1 is made of a material which transmits light in a wavelength region to be observed. For example, it is composed of inorganic compounds such as glass and quartz, polystyrene, synthetic resins, plastics such as polypropylene, polyethylene, vinyl chloride, polyphenylene ether, polyphenylene sulfide, and the like. According to this observation tool, the reflecting surface is not damaged even when the storage unit is cleaned, and the maintenance of the reflecting surface is easy.

The reflecting surface 41 can be formed by plating the material of the coupling structure 1. The plating is, for example, plating of a material having a smooth surface and a high reflectance, such as a metal such as silver.

The observation tool shown in fig. 2(a) has a reflecting surface provided between the structure 1 and the structure 40 that transmit light. The observation tool is manufactured by attaching a structure 40 having a reflection surface to the surface of the structure 1 opposite to the storage section 2. This observation tool is easy to manufacture because the structure 1 and the structure 40 are processed separately and finally joined together. The structures 1 and 40 may be bonded with an optical adhesive such as balsam or a light-curable synthetic resin adhesive. In addition, the structure 1 and the structure 40 may be used in a stacked state in observation without using an adhesive or the like. When the structure 1 and the structure 40 are used in a stacked state, they are held and fixed by a fixing tool such as a clip.

The observation tool shown in fig. 2(B) is similar to the observation tool shown in fig. 2(a), but differs in the following points: the surface 43 of the structure 40 that transmits light, which is opposite to the objective lens, is provided with a reflecting surface. The structure 1 is made of a material that transmits light, and according to this observation tool, the distance between the position of the observation target and the reflection surface can be easily adjusted by adjusting the thickness of the structure 40.

In order to use the observation tool of the present embodiment for cell observation, the culture medium is placed in the storage part together with the cells, and as shown in fig. 3(a1), the culture medium is covered with a cover glass (5 in fig. 3), and observation or detection is performed with a normal microscope using an illumination device for introducing visible light into the objective lens, or monitoring is performed using a CCD camera coupled to the objective lens. As the illumination device for introducing light, for example, japanese optical production "nikon EPI-U" or the like, which is a general illumination device, can be used.

Although fig. 3(a1) shows the case where the cover glass 5 is provided on the upper portion of the structure 1, as shown in fig. 3(a2), a method of using the structure in which the entire structure is viewed from below in reverse order may be used.

Further, the observation tool shown in fig. 3(B) and (C) may be used. The observation tool of fig. 3(B) is configured as follows: after the observation target is placed in the groove of the storage unit 2, the storage unit 2 is covered with the structure 40, and the structure 40 has a reflection surface on a surface 44 facing the objective lens.

In the cell observation device of fig. 3(C), a reflection surface is provided on a surface 45 opposite to the surface of the structure 40 that transmits light and faces the objective lens. According to this observation tool, the distance between the position of the observation target and the reflection surface can be easily changed by adjusting the thickness of the structure 40.

As shown in fig. 4(a), the observation tool of the present embodiment can be used by providing an injection port 6 for injecting an observation target and supporting the structure 1 on the cover glass 5. At this time, the observation was made from below. For example, the cells and the culture medium can be injected simultaneously through the injection port 6, and the observation operation is simple.

In addition, when observation is performed using the observation tool of the present embodiment, the observation target, for example, cells, can be observed in a floating state, for example, cells in a fluid state without adhering to the glass surface, that is, as shown in fig. 4(B) and (C), the holes 7 and 8 are provided so that the fluid can pass through the structure 1, and the state of the cells in the fluid can be observed even when the fluid containing the cells flows into one hole. For this purpose, as shown in fig. 6, a reflecting surface may be formed on the inner wall 11 of a transparent tube 12 having the cell storage part 9.

The observation tool described above can calculate cells and the like by making a scale on the storage unit as necessary as shown in fig. 5. Even in such a case, the observation tool according to the present embodiment can observe the minute transparent object with a good contrast, and therefore, calculation is easy.

When the cell observation tool of the present embodiment is used, cells, for example, blood cells such as neutrophils, eosinophils, basophils, monocytes, macrophages, and lymphocytes, or other animal cells, or plant protoplasts, or even particle components contained therein can be observed clearly by naked eyes using a general optical microscope or by observation using a monitor using a general lens and a CCD camera, and thus the cells can be observed without using a special device such as a phase-contrast microscope of the related art.

Next, as a second embodiment to which the present invention is applied, an observation method using the cell observation tool described above will be described in more detail.

The observation method of the present invention is characterized in that: a microscopic transparent object such as a cell can be observed with good contrast. Before describing the observation method according to the present invention, the principle that a minute transparent object can be observed with a good contrast by the observation method according to the present invention will be briefly described.

Fig. 17 shows a method for obtaining the contrast of an observation target image in a conventional transmission observation microscope. That is, when the incident wavefront 103 of the illumination light is transmitted through the observation target 102 in the medium 101, the outgoing wavefront 104 that exits the observation target is deformed due to the difference in refractive index between the medium 101 and the object 102. The micro-elements 105 of the emitted wavefront proceed in a direction perpendicular to the respective wavefronts. Therefore, the minute elements 105 of the outgoing wavefront that have undergone deformation travel in a direction different from the direction in which the incoming wavefront 104 travels. When a part of the minute elements 105 of the outgoing wavefront that has undergone strong deformation such as the peripheral portion of the observation target 102 goes out of the angular range indicated by the numerical aperture of the objective lens 106, a shadow is generated in a part of the image, and the observation target 102 is imaged with contrast. However, if there is almost no difference in refractive index between the object 102 and the medium 101, for example, cells in a culture medium, the minute elements 105 of the emitted wavefront that have undergone deformation also move forward within the angular range indicated by the numerical aperture of the objective lens 106, and therefore there is almost no contrast between light and dark in the object image. Therefore, it is difficult to observe an image of cells in a culture medium with a conventional transmission observation microscope.

In contrast, in the observation method of the present invention, a method of acquiring the contrast of the image of the observation target is shown in fig. 10. That is, when the object 202 close to the reflecting surface 207 is observed as epi-illumination, the incident wavefront 203 is once transmitted through the observation target 202, then reflected by the reflecting surface 207, and further transmitted through the observation target 202 again. The incident wavefront 204 formed by the incident wavefront 203 transmitting through the observation target 202 twice suffers from twice the distortion in the case of the existing transmission observation microscope. In the conventional transmission observation microscope, a part of the microscopic element 205 of the emitted wavefront advances within an angle range expressed by the numerical aperture of the objective lens 206, but if the observation method according to the present invention is used, the part can advance out of the range, and therefore a shadow is formed on the image of the observation target. Therefore, contrast of light and shade in an image can be easily obtained as compared with a conventional transmission observation microscope. Furthermore, even cells in the culture medium, which are difficult to observe with a conventional transmission observation microscope, can be clearly observed with a good contrast.

Hereinafter, a second embodiment of the present invention will be described with reference to the drawings.

Fig. 7 is a schematic view of an observation apparatus to which an embodiment of the present invention is applied. As shown in the drawing, the observation apparatus of the present embodiment includes an epi-observation microscope 311 and a culture dish 312 using a mirror coating 307 on the bottom surface.

The epi-observation microscope 311 is composed of: a lens barrel 315, an objective table 317, a light source section 313, an epi-illumination tube 314 and an objective lens 306, and a mirror base 316 supporting these together, the objective table 317 can be provided with a culture dish 312 thereon. The stage 317 is connected to the mirror base 316 and can be moved in the up-and-down direction by turning the focusing lever 318.

Fig. 8 shows a configuration of an optical system of the epi-observation microscope 311. As shown in fig. 8, the optical system is composed of: a light source 813 for generating illumination light; a collimating lens 841 for aligning the traveling direction of the illumination light emitted from the light source 813; a half mirror 842 for deflecting the traveling direction of the illumination light whose traveling direction is aligned to the vertical direction; an objective lens 806 for condensing illumination light on a specimen 808 containing an observation object while enlarging the projection specimen 808; an imaging lens 846 for imaging the specimen 808 on an imaging plate; the reflecting mirror 849 has a reflecting surface 807 that reflects the illumination light once transmitted through the specimen 808 and returns it.

The imaging lens 846 is placed in the barrel 315. The light source 813 is placed in the light source section 313, and the collimator lens 841 and the half mirror 842 are placed in the epi-illumination tube 314.

When the observation device having the above-described configuration is used to observe an object, the following is performed.

As shown in FIG. 9, a culture solution 301 is placed in a storage part of an observation tool together with an object to be observed (for example, a cell 302). A culture dish is placed on an upper surface of a stage 317. The brightness of the illumination light from the light source 313 is appropriately adjusted. The stage 317 is moved in the up-down direction by turning the focusing lever 318, and focusing and observation are performed.

In the observation method of the present embodiment, the distance d between the observation target and the reflecting surface preferably satisfies the following formula (1).

d≦W/(2NA²)…(1)

(wherein d represents the distance between the observation target and the reflecting surface, W represents the wavelength of light used for observation, and NA represents the numerical aperture of the optical system.)

The distance d between the observation target and the reflecting surface can be adjusted by adjusting the density of the medium. For example, if the viewing object is denser than the medium, the viewing object automatically settles by gravity to the bottom of the culture dish 312, near the reflecting surface there.

Further, the principle of observation with a good contrast by satisfying the above formula will be described below. When the above formula is satisfied, the observation target is disposed close to the reflection surface at a distance of about half or less of the depth of focus of the observation optical system, and the incident wavefront once transmitted through the observation target passes through the observation target again without being substantially deformed. Therefore, the image of the observation target can be observed with a good contrast.

FIG. 11 shows the results of imaging simulations using the following computational model: the calculation model was a rotational ellipsoid having a diameter of 4 μm and a height of 2 μm in the cell 302, and the refractive index of the cell 302 was 1.4 and the refractive index of the culture solution 301 was 1.33. In addition, fig. 11 is a schematic view of imaging. Here, 521 is a center line, 522 is an intensity distribution on the center line, and 523 is an image of the cell 302. The wavelength of light used for observation is 550nm in the present simulation, since the epi-illumination light passes through the cell 302 twice in a reciprocating manner, the outline of the cell 302 can be observed with a clear contrast between light and dark as shown in fig. 11(b), on the other hand, when the cell 302 under the same conditions is observed by the conventional transmission observation method, the outline of the cell 302 is not clear as shown in fig. 11 (c).

However, when the distance between the cell 302 and the mirror coating 307 is increased, the contrast of the cell image is decreased. The depth of focus Δ of the observation optical system in the present embodiment is obtained by the following equation.

Δ＝W/NA²＝0.55μm/0.452＝2.7μm

(wherein W represents the wavelength of the light used and NA represents the numerical aperture of the objective lens 306 mounted on the reflection observation microscope 311.)

That is, half of the depth of focus Δ is about 1.4 μm the results of imaging simulation using the following calculation model are shown in fig. 12(b) to (f): the calculation model is shown in FIG. 12(a), and the distances d between the cell 302 and the mirror coating 307 are (b)1 μm, (c)1.2 μm, (d)1.4 μm, (e)1.6 μm, and (f)1.8 μm, respectively. As shown, as the distance d increases from 1 μm, the contrast of the contour of the cell 302 decreases, and is hardly visible beyond 1.4 μm.

From the above, it is preferable that the distance between the cell 302 and the mirror coating 307 is about half or less of the focal depth Δ.

In the example of the present embodiment, the distance d between the observation target and the reflecting surface preferably satisfies the following formula (2).

d>F/(4tan(sin^-1NA))…(2)

(wherein d represents the distance between the observation target and the reflecting surface, F represents the diameter of the field of view of the optical system for observing the observation target, and NA represents the numerical aperture of the optical system for observing the observation target.)

When the observation tool shown in fig. 3(a2) is used, since the observation target is positioned on the cover glass 5 by gravity, the distance d between the observation target and the reflecting surface can be adjusted by adjusting the depth a of the groove of the storage unit 2 by machining.

In the case of the observation tool or the like shown in fig. 1(c), the distance between the bottom surface of the groove and the reflecting surface is adjusted by adjusting the depth of the groove of the storage portion by machining, and therefore the distance between the observation target and the reflecting surface can be adjusted. In the case of the observation tool or the like shown in fig. 2(B), the distance between the observation target and the reflecting surface can be adjusted by adjusting the thickness of the structure 40.

In addition, the distance d between the observation target and the reflecting surface can be adjusted by adjusting the density of the medium. For example, if the viewing object is less dense than the medium, the viewing object automatically moves away from the bottom of the culture dish 312 a distance from the reflecting surface there.

Further, the principle of observation with good contrast is explained below, since the above calculation formula 2 is satisfied. When the above calculation formula 2 is satisfied, the reflecting surface is away from the observation target to some extent. The numerical aperture of the illumination light is actually reduced. Therefore, the object can be observed with good contrast.

More specifically, consider the following: as shown in FIG. 13, an object 1002 is disposed at the focal position of an objective lens 1006, and a mirror 1049 having a reflecting surface 1007 is disposed at a position d away from the object 1002 to observe the object 1002 with epi-illumination, an illumination region 1009 in the vicinity of the object 1002 directly illuminated by the objective lens 1006 is limited by a diameter F by the objective lens 1006 or the specification of an optical system including the objective lens. The illumination light emitted from the objective lens 1006, transmitted through the vicinity of the object 1002 once, reflected by the reflection surface 1007, and re-irradiated to the object passes through the mirror image action of the reflection surface 1007, just as if the mirror image 1009' of the irradiation region 1009 was the light source facing the object 1002 to be irradiated. At this time, the maximum angle θ i _ max of the irradiation light viewed by the object 1002 is given by the following equation.

tanθi_max＝F/(4d)...(3)

Thus, the numerical aperture sin θ i _ max of the illumination light substantial to the object 1002 is represented by the formula

sinθi_max＝sin(tan^-1F/(4d))...(4)

It is given. This is a condition that the numerical aperture NA of the objective lens 1006 is smaller

Equation 5 is given by NA > sin θ i _ max

Namely, it is

d>F/(4tan(sin^-1NA))...(2)

The numerical aperture of the substantial illumination light is smaller than the numerical aperture of the objective lens, and the contrast of the observed image of the object 1002 is improved.

A simulation example of the situation where the observation contrast of the object is improved when the numerical aperture of the illumination light is smaller than the numerical aperture of the objective lens will be described with reference to FIG. 14, FIG. 14(a) shows a model for calculating a cell 1102 in a culture solution 1101, the cell 1102 having a diameter of 5 μm and a thickness of 2

A rotational ellipsoid of μm. The refractive index of the cells was set to 1.4, and the refractive index of the culture medium was set to 1.33. The wavelength of the illumination light was set to 550nm. The numerical aperture of the objective lens was set to 0.45. Fig. 14(b) to (f) show the following calculation examples: the numerical aperture of the illumination light is 100%, (c) 80%, (d) 60%, (e) 40%, and (f) 20% of the numerical aperture of the objective lens. It is found that as the numerical aperture of the illumination light becomes smaller than the numerical aperture of the objective lens, the contrast of the object image is improved.

Next, a minute flow path observation device to which the third embodiment of the present invention is applied will be described. The minute flow path observation device according to the third embodiment is characterized by an observation target storage unit, and is suitable for observation of cell movement.

As shown in fig. 15, the observation target storage unit has a minute flow path 612. The minute flow path 612 is constructed on the silicon substrate 631 in the present embodiment, an observation target is observed from below by an inverted epi-illumination microscope not shown, and 606 in fig. 15 denotes an objective lens.

One inlet 632 and 3 outlets 633 are provided in the minute flow path 612. The inlet 632 and the outlet 633 are connected inside the minute flow path 612 is manufactured in the following method: using semiconductor fabrication techniques, a pattern of silicon oxide 634 is formed on a silicon substrate, covering the mold with a glass plate 635 as a lid.

The thickness of the silicon oxide 634 is substantially the same as that of the cell 602. Therefore, when the cell 602 passes through the minute flow path 612, the cell 602 is almost in contact with the silicon substrate surface 607, and therefore the profile of the cell 602 can be observed with good contrast.

Further, a portion connected to the inlet 632 of the minute flow path 612 and retaining the cells is formed, so that the cells 602 flowing through the minute flow path 612 can be temporarily retained. At this time, a deep hole is formed in the silicon substrate in the cell retention portion, and thus the surface of the silicon substrate that functions as a reflection surface in this portion is separated from the glass plate 635. As described above, in the cell retention portion, the numerical aperture of the substantial illumination light is reduced, and therefore the cell 602 located in the cell retention portion can be observed with good contrast.

As shown in the drawing, an electric field generating device including a voltage control device 650, a variable voltage generating device 660, and two electrodes 665 is installed in the minute flow path 612. Two electrodes 665 are installed at the side of the minute flow path 612 to apply a voltage generated by the variable voltage device 660 electrically connected to the side of the minute flow path 612, thereby generating an electric field inside the minute flow path. The variable voltage generator 660 is electrically connected to the voltage control device, and controls the voltage generated by the variable voltage generator 660 based on a method of observing the cell 602 by an inverted epi-microscope not shown, thereby assigning the direction of the cell 602 to which of the three outlets 663 the cell 602 is going. The plurality of cells 602 flow into the minute flow path 612 sequentially from the inlet 632, and are observed by reflection by the objective lens 606. The surface 607 of the silicon substrate is used as a reflecting surface, and the cell 602 in the minute flow path 612 can be observed with good contrast. Based on this observed information, the electric field generating device transforms the electric field strength in the minute flow path 612. The direction of the flow is controlled by the internal electric field strength, so that the cells 602 in the minute flow path 612 are discharged through three outlets 633.

Thus, if the minute flow path observation device of the present embodiment is used, the minute flow path 612 can be constructed on the silicon substrate 631 by using the epi-illumination microscope as the observation means for the cell 602. Since the minute flow path on the silicon substrate 631 can use a semiconductor manufacturing technique, it can be produced in large quantities at a low cost as compared with a case where the minute flow path is constructed on an existing glass substrate. Further, compared with the conventional observation method using a special microscope, it is possible to reduce the size of the entire observation apparatus and to construct it at a low cost, such as to eliminate the need for a transmission illumination device and to simplify the stage for holding the minute flow path 612.

In the third embodiment, it is preferable that the distance between the observation target and the reflecting surface (mirror surface) satisfies the expressions (1) and (2) as described in the second embodiment.

The embodiments of the present invention have been described above.

In the above embodiment, a conventional minute transparent object which is extremely difficult to observe can be observed with a good contrast.

In addition, according to the above embodiment, since an optical system for transmission illumination is not required, there is an advantage that the entire observation device can be downsized. In addition, the following advantages are provided: even when the object is difficult to move, the object observation site can be easily adjusted by moving the entire observation device.

According to the above embodiment, by providing the reflecting surface in the container, the object can be easily brought close to the reflecting surface. In addition, a mirror is placed on the bottom of the glass vessel, and when the glass vessel is filled with a medium containing the object to be observed, the material automatically settles to the bottom of the glass vessel due to gravity, close to the reflecting surface there, if the material is denser than the medium.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the gist of the present invention.

(examples)

FIG. 18 is a photograph showing a cell photographed from below by using the observation tool shown in FIG. 3 (2A). The observation tools used were: the structure 1 was made of a silicon wafer, and the distance a between the cover glass 5 and the reflection surface was 5 μm.

A camera device: CCD digital image camera CL-211H (Watec America Co., Las Vegas, Nev)

An irradiation device: EPI-U (Nikon Kawasaki, Japan)

An objective lens: x 20

Culture solution: a culture medium containing 20mM HEPES and 0.1% bovine serum protein was added to RPMI1640 buffer.

Cell: eosinophilic leukocyte

For granulocytic differentiation in human blood, selected eosinophils were selected using a cathode through a magnetic block bound by anti-CD 16 antibody. In addition, a kinetic magnetic particle concentrator (Dynal a.s., Oslo, Norway) was used in the magnetic block, and the operation was performed according to the use method attached to the product.

As shown in fig. 18, it is clear that microscopic transparencies, i.e., acidic leukocytes and intracellular particles can be observed with good contrast.

Claims

1. An observation tool used in a method of observing an observation target by illuminating the observation target with epi-illumination light through an optical system having an objective lens, the observation tool being an observation tool for storing the observation target and comprising a structure, the observation tool comprising:

the above-mentioned structure has a tank for holding an observation target and a solution at the same time,

a reflection surface for reflecting the epi-illumination light during observation is provided on the bottom surface of the groove,

the reflecting surface is a plane.

2. An observation tool used in a method of observing an observation target by illuminating the observation target with epi-illumination light through an optical system having an objective lens, the observation tool being configured to receive and store the observation target and including a structure through which the epi-illumination light passes, the observation tool comprising:

a reflection surface for reflecting the epi-illumination light during observation is provided on a surface different from the surface on which the groove is provided,

the reflecting surface is a plane.

3. An observation method for observing an observation target by illuminating the observation target with epi-illumination light through an optical system having an objective lens by using an observation tool for storing the observation target, the observation tool being composed of a structural body, the observation method comprising:

the above-mentioned observation target is a tiny transparent body,

a reflecting surface for reflecting the epi-illumination light during observation is provided on the bottom surface of the groove,

observing the micro transparent body with the observation tool, wherein the micro transparent body is arranged at a specific distance from the reflection surface,

the reflecting surface is a plane.

4. An observation method for observing an observation target by illuminating with epi-illumination light through an optical system having an objective lens using an observation tool for storing the observation target, the observation tool being composed of a structure transmitting illumination light, the observation method comprising:

the above-mentioned observation target is a tiny transparent body,

the reflecting surface is a plane.

5. An observation method for observing an observation target by illuminating the observation target with epi-illumination light through an optical system having an objective lens by using an observation tool for storing the observation target, the observation tool being composed of a structural body, the observation method comprising:

the above-mentioned observation target is a tiny transparent body,

observing the micro transparent body directly arranged on the reflecting surface by using the observing tool,

the reflecting surface is a plane.

6. The observation method according to claim 3, wherein:

the micro-transparent body is a cell,

the liquid is a culture liquid.

7. The observation method according to claim 3, wherein:

the observation tool stores the micro transparent member so that a distance between the micro transparent member and the reflecting surface is not more than half a depth of focus of the optical system.

8. The observation method according to claim 3, wherein:

the observation tool is configured to store the micro transparent member so that a distance d between the micro transparent member and the reflection surface satisfies the following formula (1),

d≦W/(2NA²) …(1)

where d represents the distance between the observation target and the reflection surface, W represents the wavelength of light used for observation, and NA represents the numerical aperture of the optical system.

9. The observation method according to claim 3, wherein:

the observation tool stores the micro transparent member so that a numerical aperture of the illumination light to the micro transparent member is smaller than a numerical aperture of the objective lens.

10. The observation method according to claim 3, wherein:

the observation tool stores the micro transparent body so that a distance d between the micro transparent body and the reflection surface satisfies the following formula (2),

d>F/(4tan(sin^-1NA)) …(2)

wherein d represents a distance between an observation target and the reflection surface, F represents a field diameter of the optical system, and NA represents a numerical aperture of the optical system.