CN102252997B - Method for measuring refractive index of microsphere or medium and application thereof - Google Patents
Method for measuring refractive index of microsphere or medium and application thereof Download PDFInfo
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- CN102252997B CN102252997B CN201110095957.5A CN201110095957A CN102252997B CN 102252997 B CN102252997 B CN 102252997B CN 201110095957 A CN201110095957 A CN 201110095957A CN 102252997 B CN102252997 B CN 102252997B
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Abstract
The invention provides a method for measuring refractive index of a microsphere or a medium. The method comprises the following steps of: imaging the microsphere immersed in the medium with lower refractive index under parallel light irradiation; measuring the ratio of the radius R of a microsphere image to the radius r of the central bright round of the microsphere image; accurately measuring the refractive index of the microsphere under the condition of knowing the refractive index of the medium; and accurately measuring the refractive index of the medium under the condition of knowing the refractive index of the microsphere, wherein the microsphere is an optically transparent mini sphere and the medium is an optically transparent substance. The method is simple and convenient, and easy to operate. The relative refractive index can be obtained only by shooting the image of the microsphere, measuring the ratio of the radius r of the central bright round of the microsphere image to the radius R of the microsphere, and using an equation (A). The method can be used for measuring the refractive index or the concentration or the temperature change of the mixed fluid at different special points, the refractive index of a phase change critical point or the dynamic change of concentration distribution, the molecular structure and the concentration change or the temperature change in an organism and the like.
Description
Technical field
The present invention relates to optical measuring technique, particularly a kind of method and application thereof measuring microballoon or medium refraction index.
Background technology
Printing opacity microsphere particle, a kind of inductor of Chang Zuowei in many aspects, can for as the instrument carrying or check various biomedical agent in biomedical research, or as studying the model of cell and tissue optical property, for correcting the primary standard substance of scientific instrument, also can be used as electromagnetic energy carrier or sensor, and high q-factor reactor.In all these application, all need the refractive index measuring microballoon.On the other hand, determination and analysis real-time, in place for local refractive index each in nonhomogeneous media and biological living also has strong demand, because refractive index becomes certain linear ratio relation with concentration of medium and temperature usually, by measuring the transient changing of refractive index, the change about molecular composition and concentration or temperature in medium and biological living can be reflected.But be no matter the mensuration of microballoon refractive index, or in nonhomogeneous media and biological living, the mensuration of each local refractive index all acquires a certain degree of difficulty.
For the measurement of microballoon refractive index, due to its particle diameter little (being generally some tens of pm diameter), therefore can not measure by the refractive index measurement method of general solid or liquid.Existing method has several, the most frequently used is immersion method, the method is the common method of solid particle refractometry, microspheres with solid to be measured immerses in liquid by it, according to the difference presentation under the microscope of two kinds of material refractive indexes,, until find the liquid consistent with microspheres with solid refractive index, now can not there is refraction effect because both refractive indexes are identical and make microballoon no longer video picture thus indirectly judge microballoon refractive index to be measured in the liquid of continuous replacing different refractivity.The method principle is simple and clear, but has obvious limitation.One is to prepare quite a lot of different refractivity liquid, and measuring process is loaded down with trivial details.And may not necessarily find at measurement high index of refraction material the liquid matched, and some liquid also may damage microballoon, so method is subject to larger restriction.Other methods are measured by the light scattering measuring microballoon place suspending liquid with MieShi scattering scheduling theory, although this method is applicable to the microballoon of any particle diameter and refractive index, its calculate and mathematics manipulation very numerous and diverse, often can only provide approximate solution.But topmost problem measures to carry out by the microballoon all identical with refractive index to a large amount of particle diameter, and what provide is mean value, and can not measure the refractive index of single microballoon.Also report in recent years by methods such as light tweezers, but need use costliness and the equipment of complexity, and the process of mensuration is quite complicated, the accuracy of result is on the low side.Also have other method as rainbow method, digital holographic microscope method etc., all there is similar problem.That is, all can not to the refractive index of microballoon carry out easy, measure fast and accurately.
The technology that can measure local refractive index each in medium and biological living is at present very few (as fluorescence method, light tweezer method, digital holographic microscope method) especially, and all exist sample is disturbed, and because finding speed is slow and process is complicated, can only uniform dielectric be measured, and can not nonhomogeneous media be measured.Particularly require can not there be vibration or mobile to sample, otherwise just can not complete detection.And for samples such as living cells because cell and periphery medium thereof inherently can move, thus existing method is all difficult to carry out fast the inner local refractive index such as nonhomogeneous media and biological living, in real time, the Accurate Determining of original position.
Summary of the invention
The object of the invention is to overcome the shortcoming of prior art and deficiency, there is provided a kind of easy and simple to handle, quick, can measure microballoon refractive index, accurate method for measuring can be done to the inner local refractive index such as nonhomogeneous media and biological living and the parameter index such as concentration, temperature again.Described microballoon is optically transparent microspheres, can be solid-state, liquid or gaseous state; Described medium is various liquid state or gaseous state so that solid-state optical clear material.
Another object of the present invention is to the application of the method that said determination microballoon or medium refraction index and the parameter index such as concentration, temperature are provided.
Object of the present invention is achieved through the following technical solutions: a kind of method measuring microballoon or medium refraction index, comprise the steps: the microballoon be immersed in its less medium of refractive index ratio imaging under directional light irradiates, measure radius R and center thereof bright both radius of a circle r ratio of microballoon picture, can Accurate Determining microballoon refractive index in known media refractive index situation, and can the refractive index of Accurate Determining medium in known microballoon refractive index situation.
Particularly, can comprise the steps:
(1) microsphere particles is immersed in its little medium of refractive index ratio, then imaging under directional light irradiates, the bright circle of the feature of its picture to be a neighboring a be Crape ring, and the thickness of Crape ring is directly proportional to the difference of microballoon refractive index to its immersing medium refractive index;
(2) radius R and the bright radius of a circle r in center, both the calculating ratio X=r/R of microballoon picture is measured;
(3) equation is utilized:
In formula: n
1for medium refraction index; n
2for microballoon refractive index
At known media refractive index n
1when, microballoon refractive index n can be recorded
2; Or conversely, utilize known refractive index n
2microballoon, the refractive index n of its immersing medium can be recorded
1.
Described directional light irradiates the illumination light that phasecontrast microscope specifically can be adopted to provide.
The application of the method for said determination microballoon or medium refraction index, microballoon can be measured the index distribution in nonhomogeneous media or biological living as inductor, only need the position that simultaneously will measure in nonhomogeneous media or biological living to place multiple microballoon and measure.Carry out refractive index in Real-Time Monitoring or Fast Measurement medium or biological living over time time, fast camera can be adopted to carry out recording a video or taking fast, and then by exploded view picture, analyze radius R and the bright radius of a circle r in center thereof of each microballoon picture in each two field picture, calculate the refractive index of microcell residing for each microballoon.
That is the inventive method can be used for easy, to measure microballoon quickly and accurately refractive index, not only can measure the refractive index of each single microballoon, and can measure the refractive index of the multiple microballoons in same visual field simultaneously; Medium used is not limited, just can as long as this medium can not damage microballoon.Therefore different media can be used, comprise liquid and gas and even directly microballoon can be placed in air and measure.Its measurement range depends on used medium, if the refractive index of medium is n
1, then the refractive index of microballoon can be measured at n
1to n
1× 2
1/2between.Such as water is adopted to be medium (n
1=1.333), then measurable microballoon refractive index between 1.333 to 1.8856.
When adopting known index microbeads, in kind, only need to adopt the picture of quick camera system to microballoon to take, just can fast, in real time, the various medium of in-site detecting, especially the refractive index and over time of nonhomogeneous media or each microcell local of the medium under being in critical conditions, and the distribution in space.Because the refractive index of solution becomes certain linear ratio relation with the solute concentration of solution and temperature, therefore determine refractive index and also just equal to determine its material concentration or temperature.As refractive index or the concentration change of fluid-mixing space difference can be measured, the change of molecular structure and concentration in the refractive index of phase critical point or the dynamic change of CONCENTRATION DISTRIBUTION, biosome, and the thermograde and over time etc. of zones of different in sample.
Action principle of the present invention is: when microballoon is immersed in its little medium of refractive index ratio, and when being irradiated imaging by directional light, because the refraction action of optical surface to light met by microballoon, make its backlight surface marginal portion not have refract light to arrive, thus can form an edge Crape ring.Because the refractive power of microballoon changes with the difference of its refractive index with place medium refraction index, therefore the width degree of edge Crape ring is also changed thereupon.Therefore just can derived equation (A) according to relevant geometric optical theory.For microballoon provides parallel light-struck mode to have many kinds, the illumination that phasecontrast microscope provides is wherein a kind of, but is not limited thereto.
The present invention has following advantage and beneficial effect relative to prior art:
(1) method is easy, processing ease.Only need to take the image of lower microballoon and measure the ratio of microballoon inconocenter bright radius of a circle r and microsphere radius R, equation (A) just can be utilized to obtain relevant refractive index.
(2) not only can measure the refractive index of each single microballoon, and can measure the refractive index of the multiple microballoons in same visual field simultaneously.
(3) medium used is not limited, different media can be used, comprise liquid and gas and even directly microballoon can be placed in air and measure.
(4) easy, quick, Accurate Determining can be carried out to microballoon refractive index, Accurate Determining that is quick, real-time, original position can be made to the inner local refractive index such as nonhomogeneous media and biological living again.
(5) because anaclasis process completes with the light velocity, therefore in sample solution, the variations in refractive index of any moment all can cause the change of the r value in microspheres image immediately.Therefore, by High-speed Photography Technology, the Rapid Variable Design process of medium refraction index and concentration or temperature can be monitored, and measurement can not be affected because of factors such as the vibrations of the motion of sample itself or environment.
(6) little to measured medium interference.
(7) utilize micron order even submicron order particle diameter microballoon, the refractive index of micron order yardstick microcell can be measured.
(8) degree of accuracy is high, and standard deviation is only 0.19%, depends on the measuring accuracy of the bright radius of a circle r and external radius R thereof of microballoon inconocenter, improves further by adopting high-resolution camera.
Accompanying drawing explanation
Fig. 1 is that a microballoon is respectively three kinds of different liquid (a: water, b: ethanol, c: 1: 1 potpourri of glycerine and water) interior imagings.
Fig. 2 is two known (n of refractive index
2=1.4350), to be separated by the only microballoon of 165 microns, for measure glycerine in water during diffusion process with video speed (25 frames per second) clap be respectively the 3rd, the 3.21st, the 3.46th, the 3.71st and the 3.96th second several typical images (Fig. 2-1) and utilize the technology of the present invention to record two microballoons residing for the refractive index of the microcell dynamic changing process (Fig. 2-2) of the 3rd to the 4th second after glycerine drips water; In figure, t is the time, and n is refractive index, lower same.
Fig. 3 is by the known (n of a refractive index
2=1.4350) microballoon, utilizes microtubule to send into imaging (a) in a fish-egg and the change (b) of refractive index in 3 hours in the fish-egg that records with the technology of the present invention.
Fig. 4 be the refractive index of two different microcells in the NaCl miniflow in the miniature organism reactor measured with two polystyrene microspheres and concentration over time.
Fig. 5 is the NaCl aqueous solution temperature in varied situations (soft dot shown in) measured with polystyrene microsphere, shows the temperature (triangles point) of each corresponding point measured with general electronic thermometer in figure simultaneously.Both value visible quite meets.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
Take water as the refractometry of medium p-poly-phenyl ethene microballoon: by a small amount of water (n
1=1.3345) drop on a microslide, then a polystyrene microsphere is immersed in the water, under then this slide being placed in a phasecontrast microscope and by its digital imaging system imaging.Correct the space scale enlargement factor of this condition determination with image analysis software IPP after, the microballoon phase contrast image (see Fig. 1 (a)) obtained is carried out to the measurement of its radius R and the bright radius of circle r in center, obtain 2R=36.83 ± 0.06 micron respectively, 2r=25.59 ± 0.07 micron, adopts equation (1) to calculate polystyrene microsphere refractive index is 1.4330 ± 0.0002.
Embodiment 2
Take ethanol as the refractometry of medium p-poly-phenyl ethene microballoon: by a small amount of ethanol (n
1=1.3628) drop on a microslide, then a polystyrene microsphere is immersed in ethanol, under then this slide being placed in a phasecontrast microscope and by its digital imaging system imaging.Correct the space scale enlargement factor of this condition determination with image analysis software IPP after, the microballoon phase contrast image (see Fig. 1 (b)) obtained is carried out to the measurement of its radius R and the bright radius of circle r in center, obtain 2R=36.88 ± 0.06 micron respectively, 2r=31.61 ± 0.07 micron, adopts equation (1) to calculate polystyrene microsphere refractive index is 1.4321 ± 0.0002.
Embodiment 3
With glycerine and water mixed liquid for the refractometry of medium p-poly-phenyl ethene microballoon: a small amount of glycerine and water are mixed into 1: 1 mixed liquor, record its refractive index n with Abbe refractometer
1=1.4055, then mixed liquor is dropped on a microslide, then a polystyrene microsphere is immersed.Under this slide is placed in a phasecontrast microscope and by its digital imaging system imaging.Correct the space scale enlargement factor of this condition determination with image analysis software IPP after, the microballoon phase contrast image (see Fig. 1 (c)) obtained is carried out to the measurement of its radius R and the bright radius of circle r in center, obtain 2R=36.85 ± 0.06 micron respectively, 2r=34.27 ± 0.06 micron, adopts equation (1) to calculate polystyrene microsphere refractive index is 1.4359 ± 0.0002.
Embodiment 4
Take water as the refractometry of medium to glass microsphere: by a small amount of water (n
1=1.3345) drop on a slide, then a glass microsphere be immersed in the water, under then this slide being placed in the parallel light system be made up of a lens combination, and by a digital camera to imaging.Correct the space scale enlargement factor of this condition determination with image analysis software Photoshop after, the microspheres image obtained is carried out to the measurement of its radius R and the bright radius of circle r in center, obtain 2R=46.43 ± 0.19 micron respectively, 2r=28.05 ± 0.11 micron, adopts equation (1) to calculate glass microsphere refractive index is 1.5590 ± 0.0003.
Embodiment 5
With CS
2for medium is to the refractometry of reflection of electromagnetic wave medium microsphere: by a small amount of CS
2(n
1=1.6276) drop on a slide, then a reflection of electromagnetic wave medium microsphere be immersed in the water, under then this slide being placed in the parallel light system be made up of a lens combination, and by a digital camera to imaging.Correct the space scale enlargement factor of this condition determination with image analysis software Photoshop after, the microspheres image obtained is carried out to the measurement of its radius R and the bright radius of circle r in center, obtain 2R=50.12 ± 0.16 micron respectively, 2r=35.01 ± 0.12 micron, adopts equation (1) to calculate reflection of electromagnetic wave medium microsphere refractive index is 1.8225 ± 0.0002.
Embodiment 6
The refractive index Monitoring on Dynamic Change of glycerine microcell different from water mixed liquid: by a small amount of water (n
1=1.3345) drop on a slide, then two polystyrene microspheres are immersed in the water, 165 microns, the two interval.Then under this slide being placed in a phasecontrast microscope.Subsequently by a glycerine instillation water, by a numeral camera system PCO, video recording is carried out 20 seconds, to monitor the dynamic change of two polystyrene microsphere places microcell refractive index (concentration) when glycerine spreads in water with video speed (25 frames per second) to two microballoons simultaneously.Then to record under video image decompose, form 25 frames per second totally 500 frame discrete images, after two microsphere radius R in each image and the bright radius of circle r in center are measured, determine the refractive index of its place microcell with equation (1).Is microspheres image at the 3rd, the 3.21st, the 3.46th, the 3.71st and the 3.96th second respectively as shown in Fig. 2 (a), and shown in Fig. 2 (b) is the time dependent situation of refractive index of the two microballoon place microcells shown with every 40 ms intervals (speed corresponding to 25 frames/second) between the 3 to the 4 second.Because the process of refraction completes with the light velocity, therefore variations in refractive index any in sample solution all can cause the change of the r value in microspheres image immediately.Therefore, as long as the shooting speed of camera system is enough high, just can the Rapid Variable Design process of tracking and measuring solution refractive index.As adopted the image capturing apparatus of 2000 frames/second, the time dependent monitoring velocity of refractive index can be made to reach the level of frame/sub-millisecond.
Embodiment 7
In fish-egg, refractive index is monitored over time: be placed in by a Mitch fish-egg on slide and cultivate with nutrient solution, utilize microtubule technology under the microscope by the known (n of a refractive index
2=1.4350) polystyrene microsphere is sent in fish-egg, within the different time, phase contrast imaging is carried out to microballoon in fish-egg, with the change of refractive index in three hours in imaging and the fish-egg that records with aforementioned concrete steps of the present invention (1)-(3) as shown in Figure 3.As everyone knows, concentration of medium is directly proportional to its refractive index, thus according to fish-egg in variations in refractive index can know the information of the change of molecular conecentration in fish-egg.
Embodiment 8
With the polystyrene microsphere of known refractive index to the NaCl concentration change monitoring in a miniature organism reactor: by two polystyrene microsphere (n
2=1.4350) pure water (n of a bio-reactor is immersed
w=1.3330) in, 200 microns, the two interval.Then under this miniature organism reactor being placed in a phasecontrast microscope.Inject the NaCl miniflow (n of 22% again toward miniature organism reactor with a microtubule
naCl=1.371).By a high precision imaging system, video recording is carried out 20 seconds, to monitor the dynamic change of two polystyrene microsphere places microcell refractive index (concentration) when NaCl miniflow spreads in water with video speed (25 frames per second) to two microballoons simultaneously.Then to record under video image decompose, form 25 frames per second totally 500 frame discrete images, after two microsphere radius R in each image and the bright radius of circle r in center are measured, determine the refractive index n of its place microcell mixed liquor with equation (1)
m, then use equation (2):
determine the volumetric concentration M of NaCl
naCland it over time as shown in Figure 4.
Embodiment 9
The temperature of microcell local in solution is measured: the polystyrene microsphere (n by a diameter being 10 microns with the polystyrene microsphere of a known refractive index
2=1.4350) immerse the NaCl aqueous solution that is placed in a double dish, the temperature variant relation equation of refractive index (3) of this NaCl aqueous solution known is:
n
NaCl＝1.3868-at， (3)
Wherein a is the thermal refractive index coefficient of solution, and for this solution, its size is 2.68 × 10
-4, t is Celsius temperature (DEG C).And the refractive index of polystyrene microsphere between 0-60 DEG C variation with temperature very I ignore.Utilize temperature controller to change the temperature of NaCl aqueous solution, observe under phasecontrast microscope simultaneously and shoot with video-corder the phase contrast image of polystyrene microsphere, R and the r value of mensuration different temperatures hypograph, derives the refractive index n of solution with equation 1 respectively
naCl, then determine corresponding solution temperature with equation 3.It the results are shown in Figure shown in 5, visible, its value measured with measure with electronic thermometer substantially identical, degree of accuracy reach about 0.5 DEG C (as adopt high-resolution pick-up device, also can improve further).But its advantage can not produce interference because measuring to detected solution, also can the temperature of different microcell in Simultaneously test solution by placing multiple microballoon, and (this example is ten microns high temporal resolution (can reach sub-millisecond resolution with the shooting speed of 2000 frames/second) and spatial resolution, according to less microballoon, also can improve spatial resolution again).This is not attainable for general thermometer.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. one kind measures the method for microballoon or medium refraction index, it is characterized in that comprising the steps: microsphere particles to be immersed in its little medium of refractive index ratio, microballoon picture is formed under directional light irradiates, the bright circle of this microballoon similarly to be a neighboring be Crape ring, and the thickness of Crape ring is directly proportional to the difference of microballoon refractive index to its immersing medium refractive index; Measure radius R and the bright radius of a circle r in center, both the calculating ratio X=r/R of this microballoon picture, then adopt geometric optical theory to derive equation:
In formula: n
1for medium refraction index; n
2for microballoon refractive index;
According to equation (A) at known media refractive index n
1accurate Determining microballoon refractive index n in situation
2, and at known microballoon refractive index n
2the refractive index n of Accurate Determining medium in situation
1; Wherein, described microballoon is the optical clear microspheres of various form, and described medium is the optical clear material of various form.
2. the method for mensuration microballoon according to claim 1 or medium refraction index, is characterized in that: described directional light irradiates the parallel light system that specifically adopts and be made up of lens combination or phasecontrast microscope provides illumination.
3. the method for mensuration microballoon according to claim 1 or medium refraction index, is characterized in that: described microballoon is solid-state, liquid or gaseous state; Described medium is various liquid state or gaseous state so that solid-state optical clear material.
4. the application of the method for mensuration microballoon according to claim 1 or medium refraction index, microballoon is measured the index distribution in nonhomogeneous media or biological living as inductor, the position that simultaneously will measure in nonhomogeneous media or biological living is placed multiple microballoon and is measured; Carry out refractive index in Real-Time Monitoring or Fast Measurement medium or biological living over time time, fast camera is adopted to carry out recording a video or taking fast, and then by exploded view picture, analyze radius R and the bright radius of a circle r in center thereof of each microballoon picture in each two field picture, calculate the refractive index of microcell residing for each microballoon.
5. the application of the method for mensuration microballoon according to claim 4 or medium refraction index, when adopting known index microbeads, the picture of quick camera system to microballoon is adopted to take, thus the refractive index of each local of medium under measuring nonhomogeneous media or being in critical conditions and over time, and the distribution in space.
6. the application of the method for mensuration microballoon according to claim 1 or medium refraction index, measures the index distribution in nonhomogeneous media or biological living, thus determines its material concentration or temperature as inductor using microballoon.
7. the application of the method for mensuration microballoon according to claim 1 or medium refraction index, for measuring the change of molecular structure and concentration in the refractive index of fluid-mixing space difference or concentration change, the dynamic change of the refractive index of phase critical point or concentration or Temperature Distribution, biosome.
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| CN1372646A (en) * | 1999-07-30 | 2002-10-02 | 彼得·D·霍兰 | Anti-reflection coatings and coated articles |
| JP2009152307A (en) * | 2007-12-19 | 2009-07-09 | Nikon Corp | Light-emitting diode element |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1372646A (en) * | 1999-07-30 | 2002-10-02 | 彼得·D·霍兰 | Anti-reflection coatings and coated articles |
| JP2009152307A (en) * | 2007-12-19 | 2009-07-09 | Nikon Corp | Light-emitting diode element |
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| 贾光明等.用Mie氏散射理论测量聚苯乙烯微球的折射率.《光子学报》.2005,第34卷(第10期),1473-1474. * |
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