CN100498415C - Observing tool and observing method using same - Google Patents
Observing tool and observing method using same Download PDFInfo
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- CN100498415C CN100498415C CNB2004800083545A CN200480008354A CN100498415C CN 100498415 C CN100498415 C CN 100498415C CN B2004800083545 A CNB2004800083545 A CN B2004800083545A CN 200480008354 A CN200480008354 A CN 200480008354A CN 100498415 C CN100498415 C CN 100498415C
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Abstract
A transparent small object such as a cell can be simply observed without using any special modulating element needed for special observing methods. An observing tool for containing an object to be observed is used for a method for observing the object by illuminating the object with vertical illuminating light through an optical system having an objective lens. The observing tool has a reflective surface that reflects vertical illumination light when the object is observed. The reflective surface is provided on the front surface of the observing tool facing to the objective lens or on the back opposed to the front surface. The observing tool has a container holding a liquid. Using the observing tool, a cell or the like can be observed along with the culture solution.
Description
Technical field
The present invention relates to the observational technique of object, particularly relate to the observational technique of transparent small items such as cell.
Background technology
In the past, during small items such as observation of cell, used the viewed in transmittance microscope.And, in order to observe the inner structure and the form of the living cells of dye-free with high-contrast, need to use phase contrast microscope, no-output phase contrast microscope, differential interference microscope, polarization microscope etc., these microscopes possess special condenser that the phase place difference ring is arranged or differential interference prism etc., and have special lenses.
Concrete, as the method for transparent small items such as the cell in the microscopic examination nutrient solution, extensively adopting with phase difference method, deflection illumination, differential interference method etc. is the special observation (consult the spy and open flat 7-225341 communique etc.) of representative.The structure of the optical system of special observation as shown in figure 16, the structure that the clear visual field of general transmission is observed is made of following: light source 713 is used to produce illumination light; Finder lens 741 is used to make the illumination direction of light unanimity of being sent by light source 713; Catoptron 742 is used to make the working direction deflection vertical direction of the illumination light of working direction unanimity; Window lens 743 are used to gather illuminating ray; Optically focused camera lens 744 is used for shining the illumination light of having assembled to containing the sample 708 of observing object; Object lens 706 are used to enlarge projection sample 708; Imaging len 746 is used for the picture of sample 708 is imaged in imaging surface 745; Objective table 717 is used to adjust the observation place of sample 708.For this structure, add illumination modulator element 747 in the incident hole site of collector lens 744, add imaging modulator element 748 in the ejaculation hole site of object lens 706.On illumination modulator element 747, use annular slot, what use phase-plate on imaging modulator element 748 is phase difference method, what use eccentric aperture on the illumination modulator element is the deflection irradiation, and what use Polarizer and differential interference prism on illumination modulator element 747 and imaging illumination component 748 is the differential interference method.By using special observation, if different with the refractive index of on every side medium, even transparent substance also could be observed with the contrast of light and shade.Special like this observational technique, effective especially during original bio-tissue transparent, that be difficult to see with original state observed with good contrast.
But, in these special observational techniques, must be at the special modulator element of the hole site of above-mentioned collector lens 744 or object lens 706 configuration.These special modulator element prices are very high, and the multiplying power that produces object lens 706 for example when changing this modulator element also need the problem that changes etc., a lot of inconvenience are arranged in the use.
Summary of the invention
The present invention In view of the foregoing carries out, and the object of the present invention is to provide a kind of technology, the particular modulation element that does not use the special observation when observing transparent small items to need, and can observe simply.
Viewing tool of the present invention is to possess the viewing tool that the object observing of reflecting surface storage portion is arranged.In addition, this viewing tool be optical system by having object lens by the viewing tool of the object observing harvesting usefulness of using in the observational technique that falls to penetrating the illumination light illumination, object observing is observed, be provided with the above-mentioned reflecting surface that falls to penetrating illumination light of reflection when observing.
When above-mentioned reflecting surface can be arranged on and observe and opposed of above-mentioned object lens, also can be arranged on opposing face with opposed of above-mentioned object lens.
In addition, also can form the flow path of above-mentioned object observing process.In addition, the storage portion of receiving and keeping above-mentioned object observing also can possess and injects mouth that contains object observing and the mouth that makes its outflow.
In addition, observational technique of the present invention is characterised in that: it is that optical system by having object lens is by falling to penetrating the method that the illumination light illumination is observed object observing, the above-mentioned reflecting surface that falls to penetrating illumination light of reflection when observing is set in the viewing tool of the above-mentioned object observing of harvesting, in above-mentioned viewing tool, receive and keep object observing, and above-mentioned object observing is observed.
When above-mentioned reflecting surface can be arranged on and observe and on opposed of the above-mentioned object lens, also can be arranged on the opposing face with opposed of above-mentioned object lens.
In addition, above-mentioned object observing also can be the small transparent body.
In addition, above-mentioned viewing tool has the container that can keep liquid, and said vesse also can be received and kept the liquid that contains the object of observation simultaneously.At this, above-mentioned object observing also can be a cell, and aforesaid liquid also can be a nutrient solution.
In addition, also can in above-mentioned viewing tool, receive and keep above-mentioned object observing, so that the distance of above-mentioned object observing and above-mentioned reflecting surface is below half of the depth of focus of above-mentioned optical system.
Concrete, also can in above-mentioned viewing tool, receive and keep above-mentioned object observing so that above-mentioned object observing and above-mentioned reflecting surface apart from the satisfied following formula (1) of d.
d≦W/(2NA
2) …(1)
(d represents the distance of object observing and reflecting surface in the formula, and W represents used light wavelength, and NA represents the numerical aperture of optical system.)
And then, also can in above-mentioned viewing tool, take in above-mentioned object observing, so that the numerical aperture of the illumination light relative with above-mentioned object observing is littler than the numerical aperture of object lens.
Concrete, also can in above-mentioned viewing tool, take in above-mentioned object observing, so that satisfy following formula (2) apart from d between above-mentioned object observing and the above-mentioned reflecting surface.
d>F/(4tan(sin
-1NA)) …(2)
(d represents the distance between object observing and the reflecting surface in the formula, and F represents the field number of optical system, and NA represents the numerical aperture of optical system.)
If according to the present invention, optical unit that need not be special just can be with good transparent small items such as contrast observation of cell.
Promptly, use cell observation instrument of the present invention, utilize common optical microscope or use common lens and the watch-dog of ccd video camera, can pair cell, blood cell or other zooblast such as for example neutrophils, eosinocyte, basophilla white blood cell, monocyte, macrophage, lymph corpuscle, perhaps bioplast of plant etc. even the particulate component that contains clearly as, thereby pair cell is observed, and does not need to use the special device as phase contrast microscope of prior art.
Description of drawings
Fig. 1 (A)～(C) relates to the sectional view of the viewing tool of first embodiment.
Fig. 2 (A) is the sectional view with viewing tool of the structure 40 that comprises reflecting surface (B).
Fig. 3 (A1)～(C) is the sectional view with viewing tool of the lid that covers storage portion.
Fig. 4 relates to the example of other use of the viewing tool of first embodiment, (A) is the sectional view during support structure 1 on cover glass.(B) be to show the sectional view that the skeleton diagram in the time of allowing the hole that liquid passes through is set.(C) be the oblique view of the viewing tool of (B).
Fig. 5 is the top view of an example that engraves the viewing tool of scale in storage portion.
Fig. 6 (A) is the sectional view that integral body makes the viewing tool of cast.(B) be the sectional view of the dotted line AB direction of (A).
Fig. 7 is the figure of structure that shows the finder of second embodiment.
Fig. 8 is the figure of structure that shows the optical system of observation of the present invention.
Fig. 9 is the figure that shows double dish and cultured cell.
Figure 10 is the figure that shows the contrast acquisition methods of object of the present invention.
Figure 11 (a) is the figure that is presented at the computation model of the imaging simulation in second embodiment.It (c) is the figure that is expressed as the result of calculation of picture simulation that Figure 11 (b) reaches.
Figure 12 (a) is the figure of the imaging analog computation model in expression second embodiment.Figure 12 (b)～(f) is the figure of the distance of showed cell 302 and mirror coating 307 result of calculation of imaging simulation when being 1.0 μ m, 1.2 μ m, 1.4 μ m, 1.6 μ m and 1.8 μ m.
Figure 13 is the figure of reduction of the numerical aperture of the illumination light that shows that reflecting surface causes.
Figure 14 (a) shows the numerical aperture the be used for illumination light figure than the computation model of the simulation example of the object picture of the numerical aperture of object lens hour.The numerical aperture of display lighting light is the figure of result of calculation of 100%, 80%, 60%, 40% and 20% o'clock imaging simulation of the numerical aperture of objective lens during Figure 14 (b)～(f).
Figure 15 is the figure of structure of the small flow path finder of expression the 3rd embodiment.
Figure 16 is the figure of structure that shows the optical system of existing special observation.
Figure 17 is the figure of acquisition methods that shows the contrast of the microscopical object of existing viewed in transmittance.
The picture that Figure 18 is taken when being to use the viewing tool shown in Fig. 3 (A2) to observe eosinocyte.
Embodiment
Below, with reference to the description of drawings one embodiment of the present invention.At first, illustrate and relate to the viewing tool of using first embodiment of the present invention.
Viewing tool of the present invention is to be used to use microscope with viewed in reflected light and even detect the instrument of cell etc., has the part of harvesting cell etc., i.e. the object observing storage portion.And, have the face of the light of this viewing tool of reflection wavelength region may of observing at least, promptly form the face of reflecting surface.
Show an example that relates to viewing tool structure of the present invention among Fig. 1～Fig. 6.And in the drawings, arrow X represents object observing to be observed by directions X by object lens.
In the viewing tool shown in Fig. 1 (A), the 1st, the structure that constitutes by glass, synthetic resin, metal, silicon etc., the 2nd, the groove of the object observing of harvesting cell etc.The reflection of light mirror of the wavelength region may of reflection observation is set in the bottom surface 3 of storage portion 2.As the catoptron that constitutes reflecting surface, for example there are pair glass, synthetic resin, metal etc. to carry out the metal plating of silver-plated or chromium plating etc., perhaps binding metal paper tinsel and form mirror surface.In addition, for example have: material metal, silicon wafer etc., that himself can be processed into minute surface constitutes structure 1, in the man-hour that adds that groove is set, the bottom surface just directly forms catoptron, groove is set after, catoptron processing etc. is carried out in the bottom surface of groove.At this, if bottom surface 3 is that catoptron is just passable at least, the surface of structure is that catoptron is also passable all.
During with the structure 1 of silicon wafer pie graph 1 (A),, can make by the groove that forms the harvesting cell for example by common method processing silicon wafers such as mechanical lapping, chemical etchings.For example, the situation of Fig. 1 (A), catoptron 3 is mirror status by the bottom surface that makes silicon wafer and forms.And then, when the formation operation by above-mentioned groove can not fully present mirror status, as required, also can further make the bottom surface of groove form the processing of minute surface.
The size of size is that the size that can use on common microscope is just passable.For example be the grain pattern of detailed observation of cell, the degree of depth of storage portion, the preferably degree of depth of cell general alignment one deck.For example, the storage portion degree of depth is that diameter is that 1～5mm is just enough under 1～100 μ m, the circular situation, but is not limited to this, can select the size that suits as required.
In addition, as the material of structure 1, himself also can form reflecting surface by possible minute surface (reflecting surface) processing, plating etc.Can use: for example, mineral compounds such as glass, quartz; Plastic cement such as polystyrene, synthetic resin, polypropylene, tygon, vinyl chloride, polyphenylene ether, polyphenylene sulfide; Metal or alloy such as stainless steel, aluminium, bronze; Non-metal inorganic materials such as pottery.
Viewing tool shown in Fig. 1 (B) with formation such as glass, plastic cement structure 1 for example, storage portion 2 is set.And then, form the reflection horizon of the material of surface smoothings and high reflectance in the bottom 4 of storage portion 2.For example, the paper tinsel of silicon bonded wafer or film, silver-plated etc.
Viewing tool shown in Fig. 1 (C) is characterised in that: with the opposing face 41 of opposed of object lens on reflecting surface is set.Structure 1 is made of the material of the light that sees through the wavelength region may of observing.For example, constitute by plastic cement such as mineral compounds such as glass, quartz, polystyrene, synthetic resin, polypropylene, tygon, vinyl chloride, polyphenylene ether, polyphenylene sulfide etc.According to this viewing tool, even can not be damaged to reflecting surface when cleaning storage portion etc., the maintenance of reflecting surface is easy.
The material of integrated structure body 1 can form reflecting surface 41 by electroplating processing.To electroplate for example be the material that can make surface smoothing and reflectivity high, as the plating of metals such as silver.
Viewing tool shown in Fig. 2 (A) is provided with reflecting surface between structure 1 that sees through light and structure 40.This viewing tool is stained with the structure 40 that forms reflecting surface by the opposing face in the storage portion 2 of structure 1 and makes.This viewing tool is made easily, processes, makes at last it in conjunction with making to structure 1 and structure 40 respectively because be.Structure 1 and structure 40 also can bond with the optical bond of for example face cream, photo-hardening type araldite etc.In addition, do not use cementing agent etc., structure 1 and structure 40 can be piled up use during observation yet.Be piled up when using, structure 1 and structure 40 are piled up, help with setting tools such as clips and fixedly use.
Viewing tool shown in viewing tool shown in Fig. 2 (B) and Fig. 2 (A) is similar, but different on the following point: see through light structure 40, with the opposing face 43 of opposed of object lens on reflecting surface is set.Structure 1 is made of the material that sees through light.According to this viewing tool,, can easily regulate the position of object observing and the distance of reflecting surface by the thickness of adjustment structure body 40.
Be used for the observation of cell for viewing tool with present embodiment, nutrient solution and cell are together put into storage portion, shown in Fig. 3 (A1), cover with cover glass (Fig. 3 5), in object lens, use the lighting device that imports visible light, with common microscopic examination and even detection, perhaps use the ccd video camera that combines with object lens to monitor.As the lighting device of lead-in light, can use Japanese optics manufacturing " Nikon EPI-U " of selling on the market for example etc. as the general illumination device.
In addition, the situation of cover glass 5 is set on the top of structure 1 shown in Fig. 3 (A1), still, shown in Fig. 3 (A2), also can use descend on the whole reversed in order, by the below observe using method.
In addition, also can be as Fig. 3 (B) viewing tool (C).The viewing tool of Fig. 3 (B) such as following formation: after object observing being put into the groove of storage portion 2, cover storage portions 2 with structure 40, structure 40 with opposed 44 on object lens on have reflecting surface.
In addition, the cell observation instrument of Fig. 3 (C) the structure 40 that sees through light, with the opposing face 45 of opposed of object lens on reflecting surface is set.According to this viewing tool,, can easily change the position of object observing and the distance of reflecting surface by the thickness of adjustment structure body 40.
In addition, the viewing tool of present embodiment shown in Fig. 4 (A), can be provided with the inlet 6 that injects object observing, support structure 1 is used on cover glass 5.Observed by the below this moment.For example, can inject cell and nutrient solution simultaneously, observe easy and simple to handle by inlet 6.
In addition, when using the viewing tool of present embodiment to observe, object observing, for example cell do not need and the glass bonding, can observe floating state yet, for example be in the cell of flow state in liquid.That is, as Fig. 4 (B) (C) shown in, can pass through to structure 1 for making liquid, hole 7,8 is set, go into to contain the liquid of cell by an orifice flow, also can observe the state of the cell in flowing.For this purpose, as shown in Figure 6, also can on the inwall 11 of transparent pipe 12, form reflecting surface with cell storage portion 9.
The viewing tool of above-mentioned explanation as shown in Figure 5, engraves scale as required on storage portion, can carry out the calculating of cell etc.Even this kind situation can be observed small transparent substance with good contrast according to the viewing tool of present embodiment, so calculate easily.
When using the cell observation instrument of present embodiment, when using common optical microscope to observe by naked eyes, when perhaps observing by the watch-dog that uses common lens and ccd video camera, can pair cell, neutrophils for example, eosinocyte, the basophilla white blood cell, monocyte, macrophage, blood cell or other zooblasts such as lymph corpuscle, perhaps bioplast of plant etc. even the particulate component that contains clearly as, thereby pair cell is observed, and does not need to use the special device as phase contrast microscope of prior art.
Secondly, as using second embodiment of the present invention, the observational technique that has used the above-mentioned middle cell observation instrument that illustrates is described in more details.
Observational technique of the present invention is characterised in that: can observe with good small transparent substances such as contrast pair cell.At this, illustrate relate to observational technique of the present invention before, illustrate simply about can observe the principle of small transparent substance by observational technique of the present invention with good contrast.
The acquisition methods of the contrast of object observing picture as shown in figure 17 in existing viewed in transmittance microscope.That is, when illumination light incident wavefront 103 sees through object observing 102 in the medium 101,, penetrate the ejaculation wave front 104 of object observing and be out of shape because medium 101 is different with the refractive index of object 102.The small key element 105 that penetrates wave front is along advancing perpendicular to the direction of wave front separately.The small key element 105 of the ejaculation wave front of therefore, being out of shape is advanced along the direction different with incident wavefront 104 working direction.Object lens 106 form the picture of object with the wave front of certain certain angular range of being represented by numerical aperture.Periphery by object observing 102 grades and suffers very the part of the small key element 105 of the ejaculation wave front of severe deformation to break away from the angular range of representing with the numerical aperture of object lens 106 when advancing, in the part of picture, produce shade, object observing 102 imagings with contrast.But, cell in the nutrient solution etc. for example, when the refractive index of object 102 and medium 101 was almost not poor, the small key element 105 of the ejaculation wave front of being out of shape was also advanced in the angular range of representing with the numerical aperture of object lens 106, so almost there is not the contrast of light and shade in the object picture.Therefore, be difficult to observe the picture of cell in the nutrient solution with common viewed in transmittance microscope.
Relative therewith, in the observational technique of the present invention, the acquisition methods of the contrast of the picture of object observing as shown in figure 10.That is, by falling to penetrating the object 202 that illumination observation is pressed close to reflecting surface 207, incident wavefront 203 sees through object observing 202 and once and then sees through object observing 202 again by reflecting surface 207 reflections in the back.See through the distortion that incident wavefront 204 that object observing 202 forms is subjected to the twice under the existing viewed in transmittance microscope situation for twice by incident wavefront 203.In existing viewed in transmittance microscope, penetrating the part of the small key element 205 of wave front advances in the angular range of representing with the numerical aperture of object lens 206, if but according to observational technique of the present invention, can break away from this scope and advance, so on the picture of object observing, form shade.Therefore, compare the contrast that obtains light and shade in the picture easily with existing viewed in transmittance microscope.And, even the cell in the nutrient solution that is difficult to observe with common viewed in transmittance microscope also may clearly be observed with good contrast.
Below, with reference to accompanying drawing second embodiment of the present invention is described.
Fig. 7 is a skeleton diagram of using the finder of one embodiment of the present invention.As shown in the figure, the finder of present embodiment has the double dish 312 that falls to penetrating viewing microscope 311 and used mirror coating 307 in the bottom surface.
Falling to penetrating viewing microscope 311 is made of following: lens barrel 315, objective table 317, light source portion 313, fall to penetrating illumination pipe 314 and object lens 306, and the mirror pedestal 316 that supports these together.Objective table 317 can be provided with double dish 312 in the above.Objective table 317 is connected with mirror pedestal 316, can move up at upper and lower by rotating focusing handle 318.
Among Fig. 8, the structure of the optical system that falls to penetrating viewing microscope 311 is shown.As shown in Figure 8, optical system is made of following: light source 813 is used to produce illumination light; Calibrate lens 841, be used to make the working direction unanimity of the illumination light of sending by light source 813; Semi-permeable mirror 842, the working direction deflection vertical direction of the illumination light that has been used to make the working direction unanimity; Object lens 806 are used for illumination light accumulated in and contain the sample 808 of observing object, enlarge projection sample 808 simultaneously; Imaging len 846 is used to make the picture of sample 808 to become at imaging plate; Catoptron 849 has reflecting surface 807, and this reflecting surface reflecting ﹠ transmitting sample 808 illumination light is once returned it.
Use as during the finder object observing of above formation, as shown below.
As shown in Figure 9, nutrient solution 301 and object observing (for example cell 302) are put into simultaneously the storage portion of viewing tool.Double dish is arranged at above the objective table 317.Suitably regulate brightness from the illumination light of light source 313.By rotating focusing handle 318 moving stage 317 on above-below direction, focus and observe.
In the observational technique of present embodiment, object observing and above-mentioned reflecting surface preferably satisfy following formula (1) apart from d.
d≦W/(2NA
2) …(1)
(d represents the distance of object observing and reflecting surface in the formula, used light wavelength during W represents to observe, and NA represents the numerical aperture of optical system.)
Can adjusting by the density of regulating medium of object observing and reflecting surface apart from d.For example, if object observing than medium density height, object observing automatically is deposited in the bottom of double dish 312 by action of gravity, near reflecting surface herein.
And then, by satisfying above-mentioned formula, to can with good contrast observe principle be explained as follows.When satisfying above-mentioned formula, the pact of the depth of focus of the optical system of observing half with interior distance, object observing is set near reflecting surface.And, see through object observing incident wavefront once, roughly indeformable, pass through object observing again.Thus, especially going out wave front at an end-fire of object observing clearly bends.Therefore, can observe the picture of object observing with contrast well.Below, be described in detail with model.
Figure 11 illustrates and carries out the imaging Simulation result when using following computation model: this computation model as diameter 4 μ m, the ellipsoid of revolution of 2 μ m highly, is decided to be 1.4 with the refractive index of cell 302 with cell 302, and the refractive index of nutrient solution 301 is decided to be 1.33.In addition, Figure 11 is the sketch map of imaging.At this, the 521st, center line, the 522nd, the intensity distributions on the center line, the 523rd, the imaging of cell 302.The light wavelength of using in the observation is 550nm.In this simulation, fall to penetrating illumination light and see through cell 302 reciprocal twice, therefore, and can be according to shown in Figure 11 (b), with the light and shade profile of contrast observation of cell 302 clearly.On the other hand, when seeing through observational technique the cell 302 of the same terms being observed, carry out the imaging Simulation result by existing shown in Figure 11 (c), the profile of cell 302 is unintelligible.
But when the distance of cell 302 and mirror coating 307 widened, the contrast of cell picture descended.The depth of focus Δ of viewing optical system is in the present embodiment tried to achieve by following formula.
Δ＝W/NA
2＝0.55μm/0.452＝2.7μm
(W represents to use light wavelength in the formula, and NA represents to reflect the numerical aperture of the object lens of installing in the viewing microscope 311 306.)
That is, half of depth of focus Δ is about 1.4 μ m.Use the imaging Simulation result of following computation model shown in Figure 12 (b)～(f): this computation model changes the distance between cell 302 and the mirror coating 307 shown in Figure 12 (a).Be respectively (b) 1 μ m, (c) 1.2 μ m, (d) 1.4 μ m, (e) 1.6 μ m, (f) 1.8 μ m apart from d.As shown in the figure, along with distance d is begun to increase by 1 μ m, the contrast of the profile of cell 302 reduces, and almost just can not see when surpassing 1.4 μ m apart from d.
The interval of cell 302 and mirror coating 307 is preferably the pact of depth of focus Δ below half from the above.
In addition, in an example of present embodiment, object observing and reflecting surface preferably satisfy following formula (2) apart from d.
d>F/(4tan(sin
-1NA)) …(2)
(d represents the distance of object observing and reflecting surface in the formula, and F represents the field number of optical system that object observing is observed, and NA represents the numerical aperture of optical system that object observing is observed.)
When using the viewing tool shown in Fig. 3 (A2),, therefore regulate the degree of depth a of the groove of storage portion 2 by processing because the gravity object observing is positioned on the cover glass 5, can regulate object observing and reflecting surface apart from d.
In addition, the situations such as viewing tool of Fig. 1 (c), by the degree of depth of processing adjusting storage portion groove, thus the distance of the bottom surface of regulating tank and reflecting surface, so can regulate the distance of object observing and reflecting surface.The situation of the viewing tool of Fig. 2 (B) etc., the distance that can regulate object observing and reflecting surface by the thickness of adjustment structure body 40.
In addition, object observing and reflecting surface can adjust by the density of regulating medium apart from d.For example, if object observing is littler than medium density, object observing is automatically left by the bottom of double dish 312, with at the reflecting surface at this place at a distance of certain distance.
In addition, owing to satisfy aforementioned calculation formula 2, to carrying out following explanation with the principle that good contrast is observed.When satisfying aforementioned calculation formula 2, reflecting surface is the outlying observation target to a certain extent.In fact the numerical aperture of illumination light reduces.So, can observe object with good contrast.
More specifically, consider following situation: as shown in figure 13, place, the focal position configuration object 1002 at object lens 1006 disposes the catoptron 1049 with reflecting surface 1007 with object 1002, to fall to penetrating illumination observation object 1002 on the position of d.Limited by diameter F by object lens 1006 or the specification that contains by the optical system of object lens by near the irradiation area 1009 the object lens 1006 direct irradiation objects 1002.By object lens 1006 penetrate, see through object 1002 nearby once, by reflecting surface 1007 reflections, the illumination light of irradiating object is by the mirror image effect of reflecting surface 1007 once more, just as irradiation area 1009 mirror image 1009 ' becoming surface of light source shines object 1002.At this moment, the maximum angle θ i-max by the irradiates light of object 1002 is provided by following formula.
tanθi_max＝F/(4d) ...(3)
Therefore, for the numerical aperture sin θ i_max of the irradiates light of object 1002 essence by formula sin θ i_max=sin (tan
-1F/ (4d)) ... (4)
Provide.This is if the condition littler than the numerical aperture NA of object lens 1006
NA〉sin θ i_max... formula 5
Promptly
d>F/(4tan(sin
-1NA)) ...(2)
, the numerical aperture of the essence illumination light just numerical aperture than object lens is little, and the contrast of the observation picture of object 1002 is improved.
The numerical aperture of illumination light is than the numerical aperture of object lens hour, and the simulation example of the situation that the observation image contrast that calculates object is improved by Figure 14 describes.Figure 14 (a) is illustrated in the computation model of the cell 1102 in the nutrient solution 1101, and cell 1102 is as the ellipsoid of revolution of diameter 5 μ m, thickness 2 μ m.The refractive index of cell is decided to be 1.4, and the refractive index of nutrient solution is decided to be 1.33.The illumination light wavelength is decided to be 550nm.The numerical aperture of object lens is decided to be 0.45.Figure 14 (b)～(f) illustrates following calculated example: the numerical aperture of illumination light is (b) 100%, (c) 80%, (d) 60%, (e) 40%, (f) 20% of the numerical aperture of object lens.Numerical aperture along with illumination light becomes littler than the numerical aperture of object lens as can be known, and the contrast of object picture improves.
Secondly explanation is as the small flow path finder of using the 3rd embodiment of the present invention.The small flow path finder of the 3rd embodiment is a feature with the object observing storage portion, is applicable to the observation of moving of cell.
The object observing storage portion has small flow path 612 as shown in figure 15.Small flow path 612 is structured on the silicon substrate 631.In the present embodiment, falling to penetrating the illumination microscope by not shown handstand type is observed object observing by the below.Among Figure 15 606 represents object lens.
632,3 outlets 633 of an inlet are set in the small flow path 612.Inlet 632 and export 633 and be connected in small flow path 612 inside.Small flow path 612 is with following method manufacturing: use semiconductor fabrication, carry out covering this model with glass plate 635 as lid by the forming of the figure of monox 634 on silicon substrate.
In addition, the thickness of monox 634 forms the thickness roughly the same with cell 602.Therefore, cell 602 is during by small flow path 612, and cell 602 is in almost and surface of silicon 607 contacted states, so can be with the profile of good contrast observation of cell 602.
And then the part that formation is connected with the inlet 632 of this small flow path 612, cell is detained can make the cell 602 that flows in the small flow path 612 do temporary transient stop.At this moment, cell is detained the silicon substrate of part and opens a deep hole, thus, with this part as the surface of silicon substrate of reflecting surface effect away from glass plate 635.As above do, be detained in the part at this cell, the numerical aperture of the illumination light of essence reduces, and is arranged in the cell 602 that cell is detained part so can observe with good contrast.
In addition, as shown in the figure, install by voltage-operated device 650, variable voltage generating means 660 and two electric field generation devices that electrode 665 constitutes in the small flow path 612.Two electrodes 665 are installed in the side of small flow path 612, and small flow path 612 sides are applied the voltage of variable voltage device 660 generations of electrical connection, so make the inner electric field that produces of small flow path.Variable voltage generating means 660 is electrically connected with voltage-operated device, based on utilizing not shown handstand type to fall to penetrating the observational technique of microscopical cell 602, the voltage that control variable voltage generating means 660 produces distributes which direction of working direction in three outlets 663 of cell 602 to go.A plurality of cells 602 are flowed in the small flow path 612 in turn by inlet 632, reflect observation by object lens 606.Surface of silicon substrate 607 is used as reflecting surface, can observe cell 602 in the small flow path 612 with good contrast.Based on the information of this observation, the electric field intensity in the small flow path 612 of electric field generation device conversion.By the electric field intensity control working direction of inside, three outlets 633 of cell 602 differentiations in the small flow path 612 are emitted.
Like this,, used as the observation unit of cell 602 to fall to penetrating the illumination microscope, can on silicon substrate 631, make up small flow path 612 if use the small flow path finder of present embodiment.Small flow path on the silicon substrate 631 be owing to can use semiconductor fabrication, thus with existing glass substrate on make up small flow path situation compare, can be to produce in large quantities at a low price.And then, compare with the special microscopical observational technique of existing use, do not need transmission illuminator, make the objective table simplification of the small flow path 612 of maintenance etc., make the finder integral miniaturization, be configured to possibility simultaneously at a low price.
In addition, the same with what illustrate in second embodiment in the above-described 3rd embodiment, the distance of object observing and reflecting surface (minute surface) satisfies formula (1) and formula (2) for well.
More than embodiments of the present invention are illustrated.
In the above-described embodiment, can observe the existing small transparent substance that is very difficult to observe with good contrast.
In addition, if according to above-mentioned embodiment, because do not need to be used for the optical system of transillumination, so have the advantage that possibility is sought the miniaturization of finder integral body.The movement of objects situation of difficult in addition, has the following advantages: even by moving of finder integral body, can easily be adjusted the object look-out station.
If according to above-mentioned embodiment,, can easily realize the state of object in container near reflecting surface by reflecting surface is set.In addition, catoptron is set, when in this glassware, injecting the medium of the object that contains observation,,, approaches reflecting surface at this place because the action of gravity material automatically is deposited to bottom, glassware ground if material is bigger than medium density in the bottom surface of glassware.
In addition, the present invention is not limited to above-mentioned embodiment, and the various distortion in the scope of these main points all are possible.
(embodiment)
Figure 18 illustrates and uses the viewing tool shown in Fig. 3 (2A), by the photo of beneath photography cell situation.The viewing tool that uses is: structure 1 is made of silicon wafer, cover glass 5 and reflecting surface be 5 μ m apart from a.In addition, photography conditions is as follows.
Photography machine: CCD digitized video video camera CL-211H (Watec America Co., Las Vegas, Nev)
Irradiation unit: EPI-U (Nikon Kawasaki, Japan)
Object lens: * 20
Nutrient solution: use the nutrient solution that adds 20mM HEPES and 0.1% bovine serum albumin matter in the RPMI1640 damping fluid.
Cell: eosinocyte
Divide for the particle white blood cell in the human blood, use the selected eosinocyte of negative electrode selection that passes through by the magnetic block of anti-CD16 antibodies.In addition, and working power magnetic particle concentrator in the magnetic block (Dynal A.S., Oslo, Norway), according to the appended using method operation of product.
As shown in figure 18, as can be known, can with good contrast to small transparent substance, be that acid white blood cell and intracellular particle are observed.
Claims (9)
1. viewing tool, this viewing tool is used in by the optical system with object lens and throws light on by falling to penetrating illumination light, and in the method that object observing is observed, this viewing tool is by the viewing tool of the object observing harvesting usefulness that constitutes through above-mentioned first structure that falls to penetrating illumination light, it is characterized in that:
Have second structure,
Above-mentioned first structure has the groove that is used for keeping simultaneously object observing and solution,
Above-mentioned second structure is provided with the above-mentioned reflecting surface that falls to penetrating illumination light of reflection when observing,
The face different and the reflecting surface overlaid of above-mentioned second structure with the face of the groove that above-mentioned first structure is set,
Above-mentioned reflecting surface is the plane.
2. viewing tool, this viewing tool is used in by the optical system with object lens and throws light on by falling to penetrating illumination light, and in the method that object observing is observed, this viewing tool is by the viewing tool of the object observing harvesting usefulness that constitutes through above-mentioned first structure that falls to penetrating illumination light, it is characterized in that:
Have above-mentioned second structure that falls to penetrating illumination light of seeing through,
Above-mentioned first structure has the groove that is used for keeping simultaneously object observing and solution,
Above-mentioned second structure is provided with the above-mentioned reflecting surface that falls to penetrating illumination light of reflection when observing,
Face different and the face overlaid different with the reflecting surface of above-mentioned second structure with the face of the groove that above-mentioned first structure is set,
Above-mentioned reflecting surface is the plane.
3. observational technique, this observational technique is to use the viewing tool of the object observing harvesting usefulness that first structure by transmission illumination light constitutes, optical system by having object lens is by falling to penetrating the illumination light illumination and to the observational technique that object observing is observed, it is characterized in that:
Above-mentioned object observing is the small transparent body,
Above-mentioned viewing tool has second structure,
Above-mentioned first structure has the groove that is used for keeping simultaneously object observing and solution,
Above-mentioned second structure is provided with the above-mentioned reflecting surface that falls to penetrating illumination light of reflection when observing,
The face different and the reflecting surface overlaid of above-mentioned second structure with the face of the groove that above-mentioned first structure is set,
Use above-mentioned viewing tool that the above-mentioned small transparent body is observed, the above-mentioned small transparent body is arranged on apart from the specific distance of above-mentioned reflecting surface,
Above-mentioned reflecting surface is the plane.
4. observational technique, this observational technique is to use the viewing tool of the object observing harvesting usefulness that first structure by transmission illumination light constitutes, optical system by having object lens is by falling to penetrating the illumination light illumination and to the observational technique that object observing is observed, it is characterized in that:
Above-mentioned object observing is the small transparent body,
Above-mentioned viewing tool has above-mentioned second structure that falls to penetrating illumination light of seeing through,
Above-mentioned first structure has the groove that is used for keeping simultaneously object observing and solution,
Above-mentioned second structure is provided with the above-mentioned reflecting surface that falls to penetrating illumination light of reflection when observing,
Face different and the face overlaid different with the reflecting surface of above-mentioned second structure with the face of the groove that above-mentioned first structure is set.
Use above-mentioned viewing tool that the above-mentioned small transparent body is observed, the above-mentioned small transparent body is arranged on apart from above-mentioned reflecting surface specified distance,
Above-mentioned reflecting surface is the plane.
5. as the observational technique in claim 3 or 4, it is characterized in that:
The above-mentioned small transparent body is a cell,
Aforesaid liquid is a nutrient solution.
6. as the observational technique in claim 3 or 4, it is characterized in that:
The above-mentioned small transparent body of harvesting in above-mentioned viewing tool is so that the distance of the above-mentioned small transparent body and above-mentioned reflecting surface is below half of the depth of focus of above-mentioned optical system.
7. as the observational technique in claim 3 or 4, it is characterized in that:
The above-mentioned small transparent body of harvesting in above-mentioned viewing tool so that the above-mentioned small transparent body and above-mentioned reflecting surface satisfy following formula (1) apart from d,
d≦W/(2NA
2)…(1)
Wherein, d represents the distance of object observing and reflecting surface in the formula, the light wavelength of using during W represents to observe, and NA represents the numerical aperture of optical system.
8. as the observational technique in claim 3 or 4, it is characterized in that:
The above-mentioned small transparent body of harvesting in above-mentioned viewing tool is so that littler than the numerical aperture of object lens for the numerical aperture of the illumination light of the above-mentioned small transparent body.
9. as the observational technique in claim 3 or 4, it is characterized in that:
The above-mentioned small transparent body of harvesting in above-mentioned viewing tool so that the above-mentioned small transparent body and above-mentioned reflecting surface apart from the satisfied following formula (2) of d,
d>F/(4tan(sin
-1NA))…(2)
Wherein, d represents the distance of object observing and reflecting surface in the formula, and F represents the field number of optical system, and NA represents the numerical aperture of optical system.
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| Application Number | Priority Date | Filing Date | Title |
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| JP088929/2003 | 2003-03-27 | ||
| JP2003088929 | 2003-03-27 | ||
| JP207738/2003 | 2003-08-18 |
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| CNA2008101497552A Division CN101419334A (en) | 2003-03-27 | 2004-03-25 | Observing tool and observing method using same |
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| CN100498415C true CN100498415C (en) | 2009-06-10 |
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| CNB2004800083545A Expired - Fee Related CN100498415C (en) | 2003-03-27 | 2004-03-25 | Observing tool and observing method using same |
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| DE102011003569B4 (en) * | 2011-02-03 | 2013-03-21 | Leica Microsystems (Schweiz) Ag | Area light source for a transmitted light illumination device of a microscope |
| JP6984822B2 (en) | 2018-01-16 | 2021-12-22 | 浜松ホトニクス株式会社 | Interference image acquisition device and interference image acquisition method |
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2004
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