WO2013020315A1

WO2013020315A1 - Medium microballoon-based florescence correlation spectrum analysis method and device

Info

Publication number: WO2013020315A1
Application number: PCT/CN2011/079365
Authority: WO
Inventors: 匡翠方; 郝翔; 刘旭; 王婷婷
Original assignee: 浙江大学
Priority date: 2011-08-10
Filing date: 2011-09-06
Publication date: 2013-02-14
Also published as: CN102305782A

Abstract

A medium microballoon-based florescence correlation spectrum analysis method capable of being effectively applied to high concentration florescence molecular samples. Radial polarization light (R1) and tangential polarization light (R2) are used as a florescence excitation beam and a florescence suppression beam respectively, and are further applied to a florescence sample (3) and excite a florescence signal after successively being subjecting to focusing by a microscope objective (1) and the nanometric injection of a medium microballoon (2), and florescence correlation spectrum analysis is completed by collecting and analyzing the florescence signal. The corresponding devices successively include: a light source generating radial polarization light (R1) and tangential polarization light (R2), a first microscope objective (1), a medium microballoon (2), a sample shelf (5) where a florescence sample (3) is placed, and a second microscope objective (4), and further include a florescence signal analysis and processing device connected to the second microscope objective. The first microscope objective (1), medium microballoon (2), florescence sample (3) and second microscope objective (4) are all on the coaxial light path of the radial polarization light (R1) and tangential polarization light (R2), and the medium microballoon (2) is located on the object space focal plane of the first microscope objective (1).

Description

Fluorescence correlation spectrum analysis method and device based on medium microsphere

Technical field

The invention belongs to the field of optical metrology and super-resolution imaging, and in particular relates to a fluorescence correlation spectrum analysis method and device based on medium microspheres.

Background technique

The development of science and technology has greatly expanded people's horizons. With the deepening of research, people have become more and more interested in the micro-field. In many fields such as chemistry, biology, medicine, etc., in order to more accurately grasp the nature of the research object, the detection and analysis of single molecules has become an indispensable research tool. In order to meet the above requirements, the corresponding instruments and equipment have been extensively developed, and fluorescence correlation spectrum analysis devices (Fluorescence) Correlation Spectroscopy, FCS) is one of them. By dynamically recording the strength of the collected fluorescent signals and performing self-international correlation digital signal processing, FCS can effectively measure the biochemical reaction rate, molecular flow velocity and molecular diffusion in living cells in real time. It has become one of the most widely used analytical devices in biochemical research.

However, as an optical confocal imaging system, the resolution of FCS is inevitably limited by the optical far-field diffraction limit. Therefore, when the concentration of the fluorescent molecules in the test sample is too large, the single molecule measurement is often not performed efficiently and interferes with the final test result. The most straightforward way to solve this problem is to dilute the test sample. However, since many biochemical reactions must be completed in a high concentration infiltration environment, the test data obtained by dilution is not reliable, and may even be impossible due to the reaction. And the result of the test failure.

technical problem

The invention provides a fluorescence correlation spectrum analysis method and device based on medium microspheres, which use radial polarized light and tangentially polarized light respectively as a fluorescent excitation beam and a fluorescence suppression beam, and break through the optical distance through the nanojet of the medium microsphere. Limitation of field diffraction limit, improved resolution, and effective reduction of fluorescence correlation spectrum analysis device (Fluorescence) The effective excitation area in Correlation Spectroscopy (FCS) can be effectively applied to high concentration fluorescent molecular samples.

Technical solution

A fluorescence correlation spectrum analysis method based on medium microspheres, comprising the following steps:

(1) using radially polarized light as a fluorescent excitation beam, tangentially polarized light as a fluorescence suppression beam, and the fluorescence excitation beam and the fluorescence suppression beam are coaxially incident into the microscope objective in parallel, and are microscopically The objective lens is simultaneously focused on the medium microsphere; the medium microsphere is located on the object focal plane of the microscope objective, and the medium microsphere has a diameter of 1~10 Um, the refractive index is 1.4~2;

(2) the medium microspheres refocus the fluorescent excitation beam and the fluorescence suppression beam after focusing by the microscope objective in step (1), and generate nanojet on the lower surface of the dielectric microsphere; The nanojet includes a nanojet produced by a fluorescent excitation beam focused by a microscope objective in step (1) and a nanojet produced by a fluorescence suppression beam focused by a microscope objective in step (1). Wherein, the nanojet produced by the fluorescent excitation beam focused by the microscope objective in step (1) is a solid bright spot, and the nanojet produced by the fluorescence suppression beam focused by the microscope objective in step (1) is hollow. Dark spot

(3) applying the nanojet generated by the step (2) to the fluorescent sample and exciting the fluorescent signal; in other words, the nanojet generated by the fluorescent excitation beam and the nanojet generated by the fluorescence suppression beam act together on the fluorescent sample and excite Fluorescent signal

(4) The fluorescent signal excited by the step (3) is collected and analyzed to obtain a fluorescence correlation spectrum.

Wherein, the radially polarized light and the tangentially polarized light described in the step (1) may be directly incident as a working beam; or may be converted by a working beam emitted by the laser, and the conversion may be implemented by a polarization converter. It can also be implemented by other methods in the prior art.

Wherein, the microscopic objective lens described in the step (1) is preferably a large numerical aperture microscope objective lens, which may be non-immersed or immersed, wherein the non-immersed large numerical aperture microscope objective lens has a numerical aperture of 0.8 to 0.95, immersed. The numerical aperture of the large numerical aperture microscope objective lens is 1.0 to 1.4, and the magnification is 80 to 100 times.

Wherein, in step (4), the fluorescence signal excited by step (3) can be collected by a microscope objective, or can be realized by other methods in the prior art. When the fluorescent signal is collected by a microscope objective, the microscope objective may be the same microscope objective as the microscope objective described in the step (1), and the reflected fluorescent signal is collected to form a "reflection mode". Or a microscopic objective lens having the same parameters as the microscopic objective lens described in the step (1) and constituting a confocal relationship in position, at which time the transmitted fluorescent signal is collected to constitute a "transmission mode".

The analysis and processing of the collected fluorescent signals in the step (4) is implemented by a common method in the prior art, and is usually performed by using a computer.

The present invention also provides an apparatus for implementing the above-described medium microsphere-based fluorescence correlation spectrum analysis method, comprising: a light source, a first microscope objective, a medium microsphere, a sample holder and a second microscope objective, and further comprising a second microscope objective connected fluorescence signal analysis processing device; wherein

The light source for generating parallel incident coaxially polarized light and tangentially polarized light; the first microscope objective for focusing the radially polarized light and the tangentially polarized light The medium microspheres for refocusing the radially polarized light and the tangentially polarized light focused by the first microscope objective to produce a nanojet; the sample holder for placing the fluorescence to be observed a sample, the fluorescent sample is excited to generate a fluorescent signal under the action of the nanojet; the second microscope objective is used to collect the fluorescent signal; and the fluorescent signal analysis processing device is used for Analyzing and processing the collected fluorescent signals;

The first microscopic objective lens, the medium microsphere, the fluorescent sample to be observed placed on the sample holder, and the second microscopic objective lens are all located on the coaxial optical path of the radially polarized light and the tangentially polarized light. The medium microspheres are located on an object focal plane of the first microscope objective, and the medium microspheres have a diameter of 1 to 10 Um, the refractive index is 1.4~2.

Wherein, the light source may be a light source that directly emits radially polarized light and tangentially polarized light; or may be a combination of a laser light source and a polarization converter, that is, a linear light source is emitted by a laser light source and converted by a polarization converter. Radially polarized light and tangentially polarized light. The polarization converter may be any device and device that realizes the conversion of cylindrical polarized light in the prior art, preferably the polarization converter Radial-Azimuthal of ARCoptix, Sweden. Polarization Converter.

Wherein, the first microscopic objective lens and the second microscopic objective lens are preferably large numerical aperture microscopic objective lenses, which may be non-immersed or immersed, wherein the numerical aperture of the non-immersed large numerical aperture microscope objective lens is 0.8. ~0.95, the numerical aperture of the immersed large numerical aperture microscope objective lens is 1.0~1.4, and the magnification is 80~100 times.

The second microscope objective lens may be the same microscope objective as the first microscope objective lens, and the reflected fluorescent signal is collected to form a "reflection mode"; The second microscope objective can also be identical to the parameters of the first microscope objective and form a confocal relationship in position, at which time the transmitted fluorescent signal is collected to form a "transmission mode".

The fluorescent signal analysis processing device is usually a computer.

The working principle of the invention is as follows:

Radially polarized light is used as the fluorescence excitation beam, and tangentially polarized light is used as the fluorescence suppression beam, and the two beams are coaxially incident into the large numerical aperture microscope objective lens in parallel. After passing the two beams through the large numerical aperture microscope objective, a focused light field is generated near the object focus of the large numerical aperture microscope objective. Since the dielectric microspheres are located at the object focus of the large numerical aperture microscope objective, the fluorescence excitation beam and the fluorescence suppression beam will re-focus through the dielectric microspheres to produce a nanojet phenomenon on the lower surface of the dielectric microsphere due to the polarization of the two beams. The state is inconsistent, the nanojet produced by the fluorescent excitation beam is a solid bright spot, and the nanojet produced by the fluorescence suppression beam is a hollow dark spot. Due to the field limiting effect of the dielectric microspheres, the size of both nanojets will be less than the general optical far field diffraction limit in both radial and axial directions. At the same time, according to the STED principle, the effective excitation area of the FCS system will be further limited in the radial direction, and this limiting effect is continuously enhanced as the intensity of the fluorescence suppression beam increases. The reduction of the effective excitation area makes it possible that only fluorescence from a single molecule can be observed by the FCS system at the same time, thereby making single molecule observation of a high concentration fluorescent molecule sample possible, and improving the accuracy of observation. Molecules located within the effective excitation area will be excited to generate a fluorescent signal, which is further collected and analyzed by a large numerical aperture microscope objective to obtain a fluorescence correlation spectrum.

Beneficial effect

Compared with the prior art, the present invention has the following beneficial technical effects:

(1) The device of the present invention has a simple structure and is easy to implement;

(2) Breaking the optical far-field diffraction limit and increasing the resolution;

(3) The observation accuracy is high, and the scope of use is expanded, and the single-molecule observation of the high-concentration fluorescent molecular sample can be performed without diluting the sample;

(4) Low cost.

DRAWINGS

1 is a schematic view showing a first embodiment of a medium microsphere-based fluorescence correlation spectrum analyzing device of the present invention.

2 is a schematic view showing a second embodiment of a medium microsphere-based fluorescence correlation spectrum analyzing device of the present invention.

Figure 3 is a schematic illustration of radially polarized light in the present invention.

Figure 4 is a schematic illustration of tangentially polarized light in the present invention.

FIG. 5 is a schematic view showing the light intensity distribution of the nano-jet generated by the fluorescent excitation beam through the medium microspheres in the present invention.

Fig. 6 is a schematic view showing the light intensity distribution of the nano-jet produced by the fluorescence suppression beam through the medium microspheres in the present invention.

Figure 7 is a graph showing the distribution of light intensity in the radial direction (X direction) of a nanojet produced by a fluorescent excitation beam through medium microspheres of different refractive indices in the present invention.

Figure 8 is a graph showing the intensity distribution of the nano-jets in the axial direction (Z direction) of the fluorescence excitation beam generated by the medium microspheres of different refractive indices in the present invention.

Fig. 9 is a schematic view showing the distribution of light intensity in the radial direction (X direction) of the nanojet produced by the fluorescence suppression beam through the medium microspheres of different refractive indices in the present invention.

Embodiments of the invention

The invention will be described in detail below with reference to the embodiments and the drawings, but the invention is not limited thereto.

Example 1

As shown in FIG. 1 , a medium-based microsphere-based fluorescence correlation spectrum analysis apparatus includes, in order, a light source that generates radial polarized light R1 and tangentially polarized light R2, a first microscopic objective lens 1, a dielectric microsphere 2, and a sample. The rack 5 is provided with a fluorescent sample 3 on the sample holder 5, and the first microscope objective 1 is also connected to a computer (not shown in Fig. 1) for analyzing and processing the collected fluorescent signals. Wherein, the radially polarized light R1 and the tangentially polarized light R2 are coaxially and parallelly incident, and the first microscopic objective lens 1, the dielectric microsphere 2 and the fluorescent sample 3 are both in the radial polarized light R1 and the tangentially polarized light R2. On the coaxial optical path, the dielectric microsphere 2 is located on the object focal plane of the first microscope objective 1.

In the above device, the light source that generates the radially polarized light R1 and the tangentially polarized light R2 may be a light source that directly emits radially polarized light and tangentially polarized light; or may be a combination of a laser light source and a polarization converter, that is, The laser source emits linearly polarized light and is converted by a polarization converter to obtain radially polarized light and tangentially polarized light. The polarization converter may be any device and device that realizes the conversion of cylindrical polarized light in the prior art, preferably the polarization converter Radial-Azimuthal of ARCoptix, Sweden. Polarization Converter.

In the above device, the first microscope objective 1 is a large numerical aperture microscope objective, which may be non-immersed or immersed, wherein the non-immersed large numerical aperture microscope objective has a numerical aperture of 0.8 to 0.95, and the immersion large numerical aperture The numerical aperture of the microscope objective is 1.0 to 1.4, and the magnification is 80 to 100 times.

In the above device, the medium microsphere 2 has a diameter of 1 to 10 um and a refractive index of 1.4 to 2.

The fluorescence correlation spectrum analysis method using the above device is as follows:

Radially polarized light R1 is used as the fluorescent excitation beam, and tangentially polarized light R2 is used as the fluorescence suppression beam, and the two beams R1 and R2 are coaxially incident into the first microscope objective 1 in parallel.

3 and FIG. 4 are schematic diagrams of the radially polarized light R1 and the tangentially polarized light R2, respectively, it can be seen that the polarization direction of each of the radially polarized light R1 is along the radial direction, and all polarization directions constitute a divergent beam; The polarization direction of each point of the tangentially polarized light R2 is along the tangential direction, and the polarization directions of all the points constitute a vortex.

After the two beams R1 and R2 pass through the first microscope objective 1, a focused light field is generated near the object focus of the first microscope objective 1. Since the medium microsphere 2 is located at the object focus of the first microscope objective 1, the two beams R1 and R2 are simultaneously focused on the medium microsphere 2;

The medium microsphere 2 refocuses the focused two beams R1 and R2 to produce a nanojet phenomenon on the lower surface of the dielectric microsphere 2, as described in detail below:

For any form of incident light, the time domain finite element difference can be used for the light field distribution of the medium microsphere 2 (Finite Difference Time Domain (FDTD) algorithm performs accurate simulation simulation.

For the fluorescence excitation beam, since its polarization state is radial polarization R1, the calculation results show that its nanojet phenomenon appears as a solid bright spot, as shown in Fig. 5. In both the radial (X direction) and axial (Z direction) directions, the size of the spot is smaller than the optical far field diffraction limit. Further calculations show that its size is related to the refractive index of the dielectric microsphere 2 - when the refractive index of the dielectric microsphere 2 is in the range of 1.4 to 2, as the refractive index of the dielectric microsphere 2 increases, the nanojet is radially Both the (X direction) and the axial (Z direction) directions are further compressed. Figures 7 and 8 clearly describe this trend of change. Figure 7 is a graph showing the intensity distribution of the fluorescence excitation beam in the radial direction (X direction) of the nanojet produced by the medium refractive index 2 (diameter 3um) of different refractive indices; Fig. 8 is the fluorescence excitation beam passing through different refractions. The rate of the medium microspheres 2 (diameter 3um) produces a plot of the intensity distribution of the nanojet along the axial direction (Z direction).

For the fluorescence suppression beam, since its polarization state is tangentially polarized light R2, the FDTD algorithm calculation results show that its nanojet phenomenon appears as a hollow dark spot, as shown in Fig. 6. The size of the dark spot is also related to the refractive index of the medium microsphere 2, but unlike the fluorescence excitation beam, when the refractive index of the medium microsphere 2 is in the range of 1.4 to 2, the size of the dark spot does not always shrink. The radial (X-direction) dimension reaches a minimum when the refractive index is equal to 1.8, as shown in FIG. Fig. 9 is a view showing the distribution of light intensity in the radial direction (X direction) of the nanojet produced by the fluorescence suppressing beam through the dielectric microspheres 2 (diameter of 3 μm) of different refractive indices.

The main function of the fluorescent excitation beam is to excite the fluorescent molecules in the fluorescent sample 3. In fact, since the size of the nanojet produced by the fluorescent excitation beam in the above device is already smaller than the optical far field diffraction limit, a better resolution than the conventional FCS can be obtained by using only the fluorescent excitation beam. However, the effective excitation region of the FCS at this time still reaches the order of 100 nanometers in the radial direction (X direction) and the axial direction (Z direction), and single molecule analysis is still insufficient for high concentration fluorescent molecular samples, so It is necessary to further compress the effective excitation area of the FCS.

The main function of the fluorescence suppression beam is to suppress the fluorescence excitation phenomenon in the irradiation region and suppress the generation of the fluorescence signal. Since the fluorescence suppression beam is generated by the medium microsphere 2, the nanojet is a hollow dark spot, and when it is simultaneously applied to the fluorescent sample 3 by the nanojet generated by the fluorescent excitation beam, the fluorescence excitation at the center point of the spot is not affected but around it. The fluorescence excitation of the region will be suppressed, so that the fluorescence signal is only from the center point of the spot, and the purpose of compressing the effective excitation area of the FCS is achieved. The inhibition increases as the intensity of the fluorescence-suppressed beam increases, which can be expressed as:

d =1/ ( a *ς ^1/2 )

Where d is the radial (X-direction) dimension of the effective excitation area of the FCS, a is the parabolic fit coefficient, and ς is the maximum intensity I _{Sted of the} nano-jet produced by the fluorescence suppression beam through the medium microsphere 2 and the fluorescent excitation beam through the medium microsphere 2 produces the ratio of the maximum intensity I _s of the nanojet, ie

ς = I _sted / I _s .

The radial (X-direction) size of the effective excitation area of the FCS can be calculated by the above formula. For example, when the size of the dielectric microsphere 2 is 3 um and the refractive index is 1.8, the radial dimension d is approximately equal to 50 nm. The axial (Z-direction) dimension l of the effective excitation area of the FCS is equal to the axial (Z-direction) size of the nano-jet produced by the fluorescent excitation beam through the dielectric microsphere 2: such as when the size of the dielectric microsphere 2 is 3 um and the refractive index is 1.8. In time, the axial dimension l is approximately equal to 100 nm.

The fluorescence sample 3 is observed by the interaction of two nano jets and the fluorescence signal is excited. The fluorescence signal passes through the first microscope objective 1 again by reflection, and is collected by the first microscope objective 1 and connected to the first microscope objective 1 The connected computer performs analytical processing, a process called "reflection mode."

Example 2

As shown in FIG. 2, a medium-based microsphere-based fluorescence correlation spectrum analysis apparatus includes, in order, a light source that generates radial polarized light R1 and tangentially polarized light R2, a first microscopic objective lens 1, a dielectric microsphere 2, and a sample. a frame 5 and a second microscope objective 4, on which a fluorescent sample 3 is placed, and a second microscope objective 4 is connected to a computer for analyzing and processing the collected fluorescent signals (not shown in FIG. 2) ). Wherein, the radially polarized light R1 and the tangentially polarized light R2 are coaxially and parallelly incident, and the first microscope objective 1, the dielectric microsphere 2, the fluorescent sample 3 and the second microscope objective 4 are both in the radial polarization R1 and the cut. On the coaxial optical path to the polarized light R2, and the dielectric microsphere 2 is located on the object focal plane of the first microscope objective 1.

It can be seen that, compared with the apparatus of Embodiment 1, this embodiment merely adds a second microscope objective 4, and the second microscope objective 4 is identical in parameters to the first microscope objective 1. The function of the second microscope 4 is to collect the fluorescent signal generated by the fluorescent sample 3. Therefore, compared to the method of Example 1, only the steps of collecting the fluorescent signal are different. In this embodiment, the fluorescent signal will no longer enter the first microscope objective 1 by reflection, but is directly collected by the second microscope objective 4 through the fluorescent sample 3, and is connected to the computer connected to the second microscope objective 4. Analytical processing is performed, which is called "transmission mode."

In order to achieve an optimal collection effect, the second microscope objective 4 and the first microscope objective 1 form a confocal relationship in position.

Compared with the "reflection mode" in Embodiment 1, the "transmission mode" in this embodiment adds a microscope objective, thereby increasing the system cost; and, "transmission mode" is only applicable to the fluorescent sample 3 being a transparent sample. The situation, while "reflection mode" does not have this limitation.

The method and apparatus of the present invention can also be applied to the application of single molecule two-photon fluorescence correlation spectroscopy without any modification.

Claims

A fluorescence correlation spectrum analysis method based on medium microspheres, comprising the following steps:

(1) using radially polarized light as a fluorescent excitation beam, tangentially polarized light as a fluorescence suppression beam, and the fluorescence excitation beam and the fluorescence suppression beam are coaxially incident into the microscope objective in parallel, and are microscopically The objective lens is simultaneously focused on the medium microsphere; the medium microsphere is located on the object focal plane of the microscope objective, and the medium microsphere has a diameter of 1~10 Um, the refractive index is 1.4~2;

(2) the medium microspheres refocus the fluorescence excitation beam and the fluorescence suppression beam after focusing by the microscope objective in step (1), and generate nanojet on the lower surface of the medium microsphere;

(3) applying the nanojet generated by the step (2) to the fluorescent sample and exciting the fluorescent signal;

(4) The fluorescent signal excited by the step (3) is collected and analyzed to obtain a fluorescence correlation spectrum.
The medium microsphere-based fluorescence correlation spectrum analysis method according to claim 1, wherein the microscope objective of step (1) is a non-immersed large numerical aperture microscope objective or an immersed large numerical aperture microscope. The objective lens has a numerical aperture of 0.8 to 0.95, and the numerical aperture of the immersed large numerical aperture microscope objective lens is 1.0 to 1.4, and the magnification is 80 to 100 times.
The medium microsphere-based fluorescence correlation spectrum analysis method according to claim 1, wherein in step (4), the fluorescence signal excited by the step (3) is collected by a microscope objective.
The apparatus for realizing the method for analyzing a fluorescence correlation spectrum based on a medium microsphere according to any one of claims 1 to 3, comprising: a light source, a first microscope objective, a medium microsphere, a sample holder, and a a second microscope objective, further comprising a fluorescence signal analysis processing device connected to the second microscope objective; wherein

The light source for generating parallel incident coaxially polarized light and tangentially polarized light; the first microscope objective for focusing the radially polarized light and the tangentially polarized light The medium microspheres for refocusing the radially polarized light and the tangentially polarized light focused by the first microscope objective to produce a nanojet; the sample holder for placing the fluorescence to be observed a sample, the fluorescent sample is excited to generate a fluorescent signal under the action of the nanojet; the second microscope objective is used to collect the fluorescent signal; and the fluorescent signal analysis processing device is used for Analyzing and processing the collected fluorescent signals;

The first microscopic objective lens, the medium microsphere, the fluorescent sample to be observed placed on the sample holder, and the second microscopic objective lens are all located on the coaxial optical path of the radially polarized light and the tangentially polarized light. The medium microspheres are located on an object focal plane of the first microscope objective, and the medium microspheres have a diameter of 1 to 10 Um, the refractive index is 1.4~2.
The apparatus according to claim 4, wherein said first microscope objective is a non-immersed large numerical aperture microscope objective or an immersed large numerical aperture microscope objective, said non-immersed large numerical aperture The numerical aperture of the microscope objective lens is 0.8~0.95, and the numerical aperture of the immersed large numerical aperture microscope objective lens is 1.0~1.4, and the magnification is 80~100 times.
The apparatus according to claim 4, wherein said second microscope objective is a non-immersed large numerical aperture microscope objective or an immersed large numerical aperture microscope objective, said non-immersed large numerical aperture The numerical aperture of the microscope objective lens is 0.8~0.95, and the numerical aperture of the immersed large numerical aperture microscope objective lens is 1.0~1.4, and the magnification is 80~100 times.
The device of claim 4 wherein said second microscope objective is the same microscope objective as the first microscope objective.
The apparatus of claim 4 wherein said second microscope objective is identical in parameters to said first microscope objective and forms a confocal relationship in position.