CN102305782A - Method and device for analyzing fluorescent correlation spectroscopy based on medium microsphere - Google Patents
Method and device for analyzing fluorescent correlation spectroscopy based on medium microsphere Download PDFInfo
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Abstract
The invention discloses a method and device for analyzing the fluorescent correlation spectroscopy based on a medium microsphere. In the method, radial polarized light and tangential polarized light are respectively used as a fluorescent excitation beam and a fluorescent inhibition beam which are sequentially focused by a microobjective, then are subjected to nanometer spraying by virtue of the medium microsphere and then act on a fluorescent sample and excite a fluorescent signal; and fluorescent correlation spectroscopy analysis is completed by collecting and analyzing the fluorescent signal. The device sequentially comprises a light source generating the radial polarized light and the tangential polarized light, a first microobjective, the medium microsphere, a sample frame provided with the fluorescent sample and a second microobjective in sequence as well as a fluorescent signal analysis processing device connected with the second microobjective; the first microobjective, the medium microsphere, the fluorescent sample and the second microobjective are located on a coaxial optical path of the radial polarized light and the tangential polarized light; and the medium microsphere is arranged on the object focal plane of the first microobjective. The method and device provided by the invention can be effectively applied to a high-concentration fluorescent molecule sample.
Description
Technical field
The invention belongs to optical metrology and super-resolution imaging field, be specifically related to relevant spectral analysis method of a kind of fluorescence and device based on medium microsphere.
Background technology
Science and technology development makes people greatly expand the visual field of oneself.Along with going deep into of research, people have produced more and more keen interest for microscopic fields.At numerous areas such as chemistry, biology, medical science,, monomolecular detection and analysis are become a kind of requisite research means in order to grasp the character of research object more exactly.In order to satisfy above-mentioned requirements, corresponding instrument and equipment is come out by mass development, fluorescence be correlated with the analysis of spectrum device (Fluorescence Correlation Spectroscopy, FCS) just one of them.Through dynamically writing down the power of the fluorescence signal of collecting; And carry out handling from (mutually) correlated digital signals; FCS can measure information such as the biochemical reaction rate in the active somatic cell, molecule flowing velocity and molecular diffusion effectively in real time, thereby becomes one of the most widely used analytical equipment in the biochemical research.
Yet as a kind of optics confocal imaging system, the resolution characteristic of FCS has also received the restriction of optics diffraction limit of far-field inevitably.Therefore when fluorescence molecule concentration is excessive in the specimen, often can not carries out the unimolecule measurement effectively and final test result is produced interference.Addressing this problem the most directly, way is that specimen is diluted; Yet; In view of must soaking under the environment in high concentration, just can accomplish many biochemical reactions; The test data that the way of employing dilution obtains is also unreliable, even possibly can't carry out causing the result of test crash owing to reaction.
Summary of the invention
The invention provides relevant spectral analysis method of a kind of fluorescence and device based on medium microsphere; Use radial polarisation light and tangential polarization light to suppress light beam as fluorescence excitation light beam and fluorescence respectively; Nanometer through medium microsphere is sprayed; Break through the restriction of optics diffraction limit of far-field; Improve resolution; And effectively reduced relevant analysis of spectrum device (the Fluorescence Correlation Spectroscopy of fluorescence; FCS) effectively excite area in, can effectively be applied in the high concentration fluorescence molecule sample.
The relevant spectral analysis method of a kind of fluorescence based on medium microsphere may further comprise the steps:
(1) use radial polarisation light as the fluorescence excitation light beam; Tangential polarization light suppresses light beam as fluorescence; Described fluorescence excitation light beam is suppressed coaxial parallel the inciding in the microcobjective of light beam with fluorescence, and focused on the medium microsphere simultaneously by described microcobjective; Described medium microsphere is positioned on the object space focal plane of described microcobjective, and described medium microsphere diameter is 1～10um, and refractive index is 1.4～2;
(2) fluorescence excitation light beam after described medium microsphere focuses on microcobjective in step (1) and fluorescence suppress light beam and focus on once more, produce nanometer at described medium microsphere lower surface and spray (Nanojet); Described nanometer is sprayed (Nanojet) and is comprised the nanometer injection that suppresses the light beam generation by the nanometer injection of the fluorescence excitation light beam generation after the focusing of microcobjective in step (1) with by the fluorescence after the focusing of microcobjective in step (1); Wherein, The nanometer injection that is produced by the fluorescence excitation light beam after the focusing of microcobjective in step (1) is solid speck, and the nanometer injection that the fluorescence inhibition light beam after being focused on by microcobjective in step (1) produces is the blackening of hollow;
(3) the nanometer jetting action that step (2) is produced is in fluorescent samples and fluorescence excitation signal; In other words, the nanometer injection acting in conjunction that the nanometer that is produced by described fluorescence excitation light beam is sprayed and fluorescence suppresses the light beam generation is in fluorescent samples and fluorescence excitation signal;
(4) collect step (3) institute's excited fluorescent signal and carry out analyzing and processing, obtain the relevant spectrum of fluorescence.
Wherein, described radial polarisation light of step (1) and tangential polarization light can be as the direct incident of working beam; Also can be converted by the working beam that laser instrument sends, described conversion can realize through polarization converter, also can realize through additive method in the prior art.
Wherein, The described microcobjective of step (1); Be preferably large-numerical aperture micro objective; Can be non-immersion or immersion; Wherein the numerical aperture of non-immersion large-numerical aperture micro objective is 0.8～0.95; The numerical aperture of immersion large-numerical aperture micro objective is 1.0～1.4, and enlargement ratio is 80～100 times.
Wherein, can collect step (3) institute excited fluorescent signal through microcobjective in the step (4), also can realize through additive method in the prior art.When collecting described fluorescence signal through microcobjective, this microcobjective can be with the described microcobjective of step (1) be same microcobjective, collect the fluorescence signal reflect this moment, constitute " reflective-mode "; Also can be microcobjective identical and the confocal relation of formation on the position with the parameter of the described microcobjective of step (1), collect the fluorescence signal that transmission is returned this moment, constitute " transmission mode ".
Wherein, carry out analyzing and processing for the fluorescence signal of collecting in the step (4) and adopt that general mode realizes in the prior art, normally adopt computing machine to carry out analyzing and processing.
The present invention also provides the device that is used to realize the above-mentioned relevant spectral analysis method of fluorescence based on medium microsphere; Comprise successively: light source, first microcobjective, medium microsphere, specimen holder and second microcobjective also comprise the fluorescence signal APU that is connected with second microcobjective; Wherein,
Described light source is used to produce the coaxial radial polarisation light and the tangential polarization light of parallel incident; Described first microcobjective is used for described radial polarisation light and tangential polarization light are focused on; Described medium microsphere is used for radial polarisation light and tangential polarization light after focusing on through first microcobjective are focused on once more, produces nanometer and sprays; Described specimen holder is used to place fluorescent samples to be observed, and described fluorescent samples excites the generation fluorescence signal under the effect that described nanometer is sprayed; Described second microcobjective is used to collect described fluorescence signal; Described fluorescence signal APU is used to analyze and handle collected fluorescence signal;
Described first microcobjective, medium microsphere, the fluorescent samples to be observed and second microcobjective that are placed on the specimen holder all are positioned on the coaxial light path of described radial polarisation light and tangential polarization light; Described medium microsphere is positioned on the object space focal plane of described first microcobjective; Described medium microsphere diameter is 1～10um, and refractive index is 1.4～2.
Wherein, described light source can be for directly sending the light source of radial polarisation light and tangential polarization light; Also can be the combination of LASER Light Source and polarization converter, that is, send the line polarisation and be converted to radial polarisation light and tangential polarization light through polarization converter by LASER Light Source.Described polarization converter can be preferably the polarization converter Radial-Azimuthal Polarization Converter of Sweden ARCoptix company for realizing any device and the device of the conversion of cylindrical polarized light in the prior art.
Wherein, Described first microcobjective and second microcobjective; Be preferably large-numerical aperture micro objective; Can be non-immersion or immersion; Wherein the numerical aperture of non-immersion large-numerical aperture micro objective is 0.8～0.95; The numerical aperture of immersion large-numerical aperture micro objective is 1.0～1.4, and enlargement ratio is 80～100 times.
Described second microcobjective can be same microcobjective with described first microcobjective, collects the fluorescence signal that reflects this moment, constitutes " reflective-mode "; Described second microcobjective also can be identical with the parameter of described first microcobjective, and on the position, constitute confocal relation, collects the fluorescence signal that transmission is returned this moment, constitutes " transmission mode ".
Described fluorescence signal APU is generally computing machine.
Principle of work of the present invention is following:
Use radial polarisation light as the fluorescence excitation light beam, tangential polarization light suppresses light beam as fluorescence, coaxial parallel the inciding in the large-numerical aperture micro objective of two light beams.Two light beams produce near the focus in object space of large-numerical aperture micro objective and focus on light field through behind the large-numerical aperture micro objective.Because medium microsphere is positioned on the focus in object space of large-numerical aperture micro objective; Therefore fluorescence excitation light beam and fluorescence inhibition light beam will be through the focussing force once more of medium microsphere; Produce the nanometer jet phenomenon at the medium microsphere lower surface; Because two light beam polarization states are inconsistent; The nanometer that the fluorescence excitation light beam produces is sprayed and is solid speck, and the nanometer that fluorescence inhibition light beam produces is sprayed and is the blackening of hollow.Because the field restriction effect of medium microsphere, the size that two kinds of nanometers are sprayed all will be less than general optics diffraction limit of far-field on radial and axial.Simultaneously, according to the STED principle, the area that effectively excites of FCS system will be diametrically by further restriction, and this restriction effect suppresses the increase of light beam light intensity along with fluorescence and constantly strengthens.Effectively excite area reduce to make at synchronization, only possibly arrived by the FCS systematic observation from monomolecular fluorescence, become possibility thereby the unimolecule of high concentration fluorescence molecule sample is observed, improved the accuracy of observing.Be positioned at the molecule that effectively the excites area generation fluorescence signal that will be excited, further collect and analyzing and processing, obtain the relevant spectrum of fluorescence through large-numerical aperture micro objective.
Than prior art, the present invention has following beneficial technical effects:
(1) apparatus of the present invention is simple in structure, and implementation method and principle are easy;
(2) break the restriction of optics diffraction limit of far-field, improved resolution;
(3) observe the accuracy height, usable range enlarges to some extent, can under the prerequisite of sample not being diluted, carry out unimolecule to high concentration fluorescence molecule sample and observe;
(4) with low cost.
Description of drawings
Fig. 1 is the synoptic diagram of first kind of embodiment of the relevant analysis of spectrum device of the fluorescence based on medium microsphere of the present invention.
Fig. 2 is the synoptic diagram of second kind of embodiment of the relevant analysis of spectrum device of the fluorescence based on medium microsphere of the present invention.
Fig. 3 is the synoptic diagram of radial polarisation light among the present invention.
Fig. 4 is the synoptic diagram of tangential polarization light among the present invention.
The light distribution synoptic diagram that Fig. 5 sprays for the nanometer that fluorescence excitation light beam among the present invention produces via medium microsphere.
Fig. 6 suppresses the light distribution synoptic diagram that light beam sprays via the nanometer of medium microsphere generation for fluorescence among the present invention.
Fig. 7 sprays the radially curve of light distribution figure of (directions X) for fluorescence excitation light beam among the present invention via the nanometer of the medium microsphere generation of different refractivity.
Fig. 8 sprays the curve of light distribution figure of (Z direction) vertically for fluorescence excitation light beam among the present invention via the nanometer of the medium microsphere generation of different refractivity.
Fig. 9 sprays the radially light distribution synoptic diagram of (directions X) for fluorescence among the present invention suppresses light beam via the nanometer of the medium microsphere generation of different refractivity.
Embodiment
Describe the present invention in detail below in conjunction with embodiment and accompanying drawing, but the present invention is not limited to this.
As shown in Figure 1; The relevant analysis of spectrum device of a kind of fluorescence based on medium microsphere; Comprise successively: the light source, first microcobjective 1, medium microsphere 2 and the specimen holder 5 that produce radial polarisation light R1 and tangential polarization light R2; Be placed with fluorescent samples 3, the first microcobjectives 1 on the specimen holder 5 and also be connected with the computing machine (at Fig. 1 and not shown) that is used for analyzing and handling collected fluorescence signal.Wherein, Radial polarisation light R1 and parallel incident coaxial with tangential polarization light R2; First microcobjective 1, medium microsphere 2 and fluorescent samples 3 threes all are on the coaxial light path of radial polarisation light R1 and tangential polarization light R2, and medium microsphere 2 is positioned on the object space focal plane of first microcobjective 1.
In the said apparatus, produce the light source of radial polarisation light R1 and tangential polarization light R2, can be for directly sending the light source of radial polarisation light and tangential polarization light; Also can be the combination of LASER Light Source and polarization converter, that is, send the line polarisation and be converted to radial polarisation light and tangential polarization light through polarization converter by LASER Light Source.Described polarization converter can be preferably the polarization converter Radial-Azimuthal Polarization Converter of Sweden ARCoptix company for realizing any device and the device of the conversion of cylindrical polarized light in the prior art.
In the said apparatus; First microcobjective 1 is a large-numerical aperture micro objective; Can be non-immersion or immersion; Wherein the numerical aperture of non-immersion large-numerical aperture micro objective is 0.8～0.95; The numerical aperture of immersion large-numerical aperture micro objective is 1.0～1.4, and enlargement ratio is 80～100 times.
In the said apparatus, the diameter of medium microsphere 2 is 1～10um, and refractive index is 1.4～2.
It is following that the employing said apparatus carries out the relevant spectral analysis method of fluorescence:
Use radial polarisation light R1 as the fluorescence excitation light beam, tangential polarization light R2 suppresses light beam as fluorescence, two light beam R1 and coaxial parallel the inciding in first microcobjective 1 of R2.
Fig. 3 and Fig. 4 are respectively the synoptic diagram of radial polarisation light R1 and tangential polarization light R2, and visible, the polarization direction that radial polarisation light R1 is every all is along radial direction, and all polarization directions constitute an angular-spread beam; And the polarization direction of every of tangential polarization light R2 all is along tangential direction, and the polarization direction of being had a few constitutes a vortex.
Two light beam R1 and R2 produce near the focus in object space of first microcobjective 1 and focus on light field through behind first microcobjective 1.Because medium microsphere 2 is positioned on the focus in object space of first microcobjective 1, therefore, two light beam R1 and R2 are focused on the medium microsphere 2 simultaneously;
Two light beam R1 and R2 after 2 pairs of focusing of medium microsphere focus on once more, produce the nanometer jet phenomenon at medium microsphere 2 lower surfaces, describe in detail as follows:
For the incident light of arbitrary form, (Finite Difference Time Domain, FDTD) algorithm carries out accurate analogue simulation to it can to use time domain finite element difference through the optical field distribution of medium microsphere 2.
For the fluorescence excitation light beam, because its polarization state is radial polarisation light R1, result of calculation shows that its nanometer jet phenomenon is rendered as a solid speck, as shown in Figure 5.At radially (directions X) with axially on (Z direction) both direction, the size of hot spot is all less than the optics diffraction limit of far-field.Further calculate and show; Its size is relevant with the refractive index of medium microsphere 2---when the refractive index of medium microsphere 2 is in 1.4～2 intervals; Along with the increase of medium microsphere 2 refractive indexes, nanometer is injected in radially on (directions X) and axial (Z direction) both direction all by further compression.Fig. 7 and Fig. 8 have clearly described this variation tendency.Be illustrated in figure 7 as the fluorescence excitation light beam and spray the radially curve of light distribution figure of (directions X) via the nanometer of medium microsphere 2 (diameter the is 3um) generation of different refractivity; Fig. 8 sprays the curve of light distribution figure of (Z direction) vertically for the fluorescence excitation light beam via the nanometer of medium microsphere 2 (diameter the is 3um) generation of different refractivity.
Suppress light beam for fluorescence, because its polarization state is tangential polarization light R2, FDTD algorithm computation result shows that its nanometer jet phenomenon is rendered as a hollow blackening, as shown in Figure 6.The size of the blackening also refractive index with medium microsphere 2 is relevant; But it is different with the fluorescence excitation light beam; When the refractive index of medium microsphere 2 is in 1.4～2 intervals; The size of blackening does not present the trend of always dwindling; Its radially (directions X) size equals to reach minimal value at 1.8 o'clock in refractive index, as shown in Figure 9.Fig. 9 sprays the radially light distribution synoptic diagram of (directions X) for fluorescence suppresses light beam via the nanometer of medium microsphere 2 (diameter the is 3um) generation of different refractivity.
The main effect of fluorescence excitation light beam is that the fluorescence molecule in the fluorescent samples 3 is excited.In fact because the nanometer that in said apparatus, is produced by the fluorescence excitation light beam is sprayed size less than the optics diffraction limit of far-field, only use the fluorescence excitation light beam just can obtain better resolution than traditional F CS.However; The effective excitation area of FCS of this moment still reaches hundred nanometer scale in radially (directions X) and size on axial (Z direction); It is still not enough for high concentration fluorescence molecule sample, to carry out single molecule analysis, therefore is necessary effectively to excite area further to compress to FCS.
The main effect that fluorescence suppresses light beam is the fluorescence excitation phenomenon that suppresses irradiation area, the generation of compacting fluorescence signal.Because it is the blackening of hollow through the nanometer injection that medium microsphere 2 produces that fluorescence suppresses light beam; The nanometer that itself and fluorescence excitation light beam produce is sprayed and is acted on 3 last times of fluorescent samples simultaneously; Unaffected but the fluorescence excitation its peripheral region of the fluorescence excitation at spot center point place will be pressed; Thereby make fluorescence signal only come from the spot center point, reach the purpose that compression FCS effectively excites area.Inhibiting effect suppresses the increase of light beam light intensity along with fluorescence and strengthens, and can use formulae express to be:
Wherein d is radially (directions X) size that FCS effectively excites area, and a is the fitting of parabola coefficient, and ζ is that fluorescence suppresses the largest light intensity I that light beam produces the nanometer injection through medium microsphere 2
Sted Cross medium microsphere 2 with the fluorescence excitation light beam and produce the largest light intensity I that nanometer is sprayed
sRatio, promptly
ζ＝I
sted/I
s
Can calculate radially (directions X) size that FCS effectively excites area through above-mentioned formula, as the size when medium microsphere 2 be 3um, when refractive index is 1.8, radial dimension d approximates 50nm.Axial (Z direction) size l that FCS effectively excites area and fluorescence excitation light beam produce axial (Z direction) size that nanometers spray through medium microsphere 2 and equate: as the size when medium microsphere 2 be 3um, when refractive index is 1.8, axial dimension l approximates 100nm.
Acting in conjunction through two kinds of nanometers are sprayed is observed and the fluorescence excitation signal fluorescent samples 3; Fluorescence signal is through reflecting once more through first microcobjective 1; Collect and insert the computing machine that is connected with first microcobjective 1 by first microcobjective 1 and carry out analyzing and processing, this process is called as " reflective-mode ".
As shown in Figure 2; The relevant analysis of spectrum device of a kind of fluorescence based on medium microsphere; Comprise successively: the light source, first microcobjective 1, medium microsphere 2, specimen holder 5 and second microcobjective 4 that produce radial polarisation light R1 and tangential polarization light R2; Be placed with fluorescent samples 3, the second microcobjectives 4 on the specimen holder 5 and also be connected with the computing machine (at Fig. 2 and not shown) that is used for analyzing and handling collected fluorescence signal.Wherein, Radial polarisation light R1 and parallel incident coaxial with tangential polarization light R2; First microcobjective 1, medium microsphere 2, fluorescent samples 3 and second microcobjective 4 all are on the coaxial light path of radial polarisation light R1 and tangential polarization light R2, and medium microsphere 2 is positioned on the object space focal plane of first microcobjective 1.
It is thus clear that, compare with the device of embodiment 1, present embodiment just increased by one second microcobjective, 4, the second microcobjectives 4 and first microcobjective, 1 parameter identical.The effect of second microscope 4 is to collect the fluorescence signal that is produced by fluorescent samples 3.Therefore, comparing with the method for embodiment 1, only is different in the step of collecting fluorescence signal.In the present embodiment; Fluorescence signal will no longer get into first microcobjective 1 through reflection once more; Collected by second microcobjective 4 but directly see through fluorescent samples 3, and insert the computing machine that second microcobjective 4 connects and carry out analyzing and processing, this process is called as " transmission mode ".
In order to reach best collecting effect, second microcobjective 4 and first microcobjective 1 constitute confocal relation on the position.
Compare with " reflective-mode " among the embodiment 1, " transmission mode " in the present embodiment increased a microcobjective, therefore improved system cost; And " transmission mode " only is applicable to that fluorescent samples 3 is the situation of transparent sample, and " reflective-mode " then do not have this restriction.
The inventive method and device also go for the application of the relevant analysis of spectrum of unimolecule two-photon fluorescence under the situation of not doing any change.
Claims (8)
1. the relevant spectral analysis method of the fluorescence based on medium microsphere is characterized in that, may further comprise the steps:
(1) use radial polarisation light as the fluorescence excitation light beam; Tangential polarization light suppresses light beam as fluorescence; Described fluorescence excitation light beam is suppressed coaxial parallel the inciding in the microcobjective of light beam with fluorescence, and focused on the medium microsphere simultaneously by described microcobjective; Described medium microsphere is positioned on the object space focal plane of described microcobjective, and described medium microsphere diameter is 1～10um, and refractive index is 1.4～2;
(2) fluorescence excitation light beam after described medium microsphere focuses on microcobjective in step (1) and fluorescence suppress light beam and focus on once more, produce nanometer at described medium microsphere lower surface and spray;
(3) the nanometer jetting action that step (2) is produced is in fluorescent samples and fluorescence excitation signal;
(4) collect step (3) institute's excited fluorescent signal and carry out analyzing and processing, obtain the relevant spectrum of fluorescence.
2. the relevant spectral analysis method of the fluorescence based on medium microsphere as claimed in claim 1; It is characterized in that; The described microcobjective of step (1) is non-immersion large-numerical aperture micro objective or immersion large-numerical aperture micro objective; The numerical aperture of described non-immersion large-numerical aperture micro objective is 0.8～0.95; The numerical aperture of described immersion large-numerical aperture micro objective is 1.0～1.4, and enlargement ratio is 80～100 times.
3. the relevant spectral analysis method of the fluorescence based on medium microsphere as claimed in claim 1 is characterized in that, in the step (4), collects step (3) institute excited fluorescent signal through microcobjective.
4. be used to realize device like the arbitrary described relevant spectral analysis method of fluorescence based on medium microsphere of claim 1～3; It is characterized in that; Comprise successively: light source, first microcobjective, medium microsphere, specimen holder and second microcobjective also comprise the fluorescence signal APU that is connected with second microcobjective; Wherein,
Described light source is used to produce the coaxial radial polarisation light and the tangential polarization light of parallel incident; Described first microcobjective is used for described radial polarisation light and tangential polarization light are focused on; Described medium microsphere is used for radial polarisation light and tangential polarization light after focusing on through first microcobjective are focused on once more, produces nanometer and sprays; Described specimen holder is used to place fluorescent samples to be observed, and described fluorescent samples excites the generation fluorescence signal under the effect that described nanometer is sprayed; Described second microcobjective is used to collect described fluorescence signal; Described fluorescence signal APU is used to analyze and handle collected fluorescence signal;
Described first microcobjective, medium microsphere, the fluorescent samples to be observed and second microcobjective that are placed on the specimen holder all are positioned on the coaxial light path of described radial polarisation light and tangential polarization light; Described medium microsphere is positioned on the object space focal plane of described first microcobjective; Described medium microsphere diameter is 1～10um, and refractive index is 1.4～2.
5. device as claimed in claim 4; It is characterized in that; Described first microcobjective is non-immersion large-numerical aperture micro objective or immersion large-numerical aperture micro objective; The numerical aperture of described non-immersion large-numerical aperture micro objective is 0.8～0.95; The numerical aperture of described immersion large-numerical aperture micro objective is 1.0～1.4, and enlargement ratio is 80～100 times.
6. device as claimed in claim 4; It is characterized in that; Described second microcobjective is non-immersion large-numerical aperture micro objective or immersion large-numerical aperture micro objective; The numerical aperture of described non-immersion large-numerical aperture micro objective is 0.8～0.95; The numerical aperture of described immersion large-numerical aperture micro objective is 1.0～1.4, and enlargement ratio is 80～100 times.
7. device as claimed in claim 4 is characterized in that, described second microcobjective and first microcobjective are same microcobjective.
8. device as claimed in claim 4 is characterized in that, the parameter of described second microcobjective and first microcobjective is identical, and on the position, constitutes confocal relation.
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| CN201110228435A CN102305782A (en) | 2011-08-10 | 2011-08-10 | Method and device for analyzing fluorescent correlation spectroscopy based on medium microsphere |
| PCT/CN2011/079365 WO2013020315A1 (en) | 2011-08-10 | 2011-09-06 | Medium microballoon-based florescence correlation spectrum analysis method and device |
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