CN110907414A - Two-dimensional sub-ten-nanometer positioning method and device based on parallel detection - Google Patents
Two-dimensional sub-ten-nanometer positioning method and device based on parallel detection Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Abstract
The invention discloses a two-dimensional sub-ten nanometer positioning method based on parallel detection, which comprises the steps of exciting one beam of hollow spot, quenching the fluorescence of the other beam of hollow spot to obtain a dark spot model exceeding the diffraction limit, sampling and counting the number of fluorescence photons at different spatial position points based on a detector array, and accurately measuring the fluorescent molecule positioning under the condition of high-concentration dye according to a mathematical model estimated by a statistical distribution rule and a maximum likelihood probability. Compared with the traditional method, the invention can reduce the positioning range, improve the concentration of dye molecules by one order of magnitude, realize the positioning of single molecules by only one-time illumination based on the principle of parallel differential detection, and greatly improve the positioning speed, thereby better meeting the actual requirements in the field of biomedical research. Meanwhile, the invention also discloses a two-dimensional sub-ten nanometer positioning device based on parallel detection.
Description
Technical Field
The invention belongs to the field of super-resolution, and particularly relates to a two-dimensional sub-ten-nanometer positioning method and device based on parallel detection.
Background
In the prior art, a paper is published by Stefan Hell in the journal of Science by Nobel chemical prize [ Nanometer resolution imaging and tracking of fluorescent molecules with a minor photon fluxes. Science 355(6325), 606-.
Different from the random coordinate positioning technology which adopts Gaussian center fitting positioning and the determined coordinate resolution technology which adopts a hollow light beam to carry out fluorescence loss, in the single molecule positioning process, the MINLUX only adopts the hollow light beam to excite fluorescence and carries out the dark spot center positioning for determining coordinates.
Compared with Gaussian center positioning, the dark spot center positioning adopted by the MINLUX has strong photosensitivity, and the spatial resolution capability of the MINLUX can reach sub-ten nanometers.
The number of photons required by the MINFUX for positioning a single fluorescent molecule is extremely low, so that the risk of bleaching fluorescent dye is greatly reduced, and the fluorescent molecule can be positioned for a long time. Therefore, the technology is a positioning means with long time, high precision and high resolution.
Although minfilx achieved sub-ten nanometer accurate localization at low luminous flux and was experimentally verified, the following disadvantages still remain:
(1) in the diffraction limit range (200 nm), if more than one fluorescent molecule appears in a certain area, the positioning precision of a single molecule is reduced by the influence of different molecules, so that the method can only exert the high-precision characteristic in the dye molecule environment with sparse concentration.
(2) When single-molecule positioning is carried out, the position of a fluorescent molecule can be obtained only by illuminating the mobile platform for four times, and as a time-sharing positioning technology, the positioning speed is limited to a certain extent.
Disclosure of Invention
The invention provides a two-dimensional sub-ten nanometer positioning method based on parallel detection, which utilizes a super-diffraction dark spot excited by one beam of hollow spot and quenched by the fluorescence of the other beam of hollow spot, adopts a detector array and combines the post mathematical model processing to realize the accurate positioning of fluorescent molecules in a high-concentration dye molecule environment.
A two-dimensional sub-ten nanometer positioning method based on parallel detection comprises the following steps:
1) the exciting light is converged into a hollow light spot to illuminate the sample, so that the sample is in an exciting state;
2) the lost light is converged into a hollow light spot illumination sample, and the hollow light spot illumination sample is superposed with the center of the hollow light spot of the exciting light to excite the fluorescent molecules to emit signal light;
3) and collecting the signal light, and acquiring an image at a corresponding scanning position and position information of the fluorescent molecules through the received signal light.
In this application, the centers of two hollow faculas that excitation light and loss light are gathered must absolutely align, and after the illumination of loss light, except excitation light center, the excited state electron of the rest part returns the ground state through the mode of stimulated radiation to different extents.
Preferably, in step 3), the signal light is received by a detector array including a plurality of single detectors, and each single detector receives a different proportion of the number of photons.
The traditional photon receiving method only adopts a single photon detection counter to position a fluorescent molecule, and the light spot needs to be accurately scanned four times, so that the center of the dark spot is respectively positioned at the center of a detection circle and at the trisection position on the circumference. The photon distribution generated at four positions after the fluorescent molecule is excited satisfies the polynomial distribution. However, this positioning technique requires accurate scanning of light spots on the nanoscale, and is slow in speed and low in efficiency for realizing one-time positioning. According to the invention, a single detector is replaced by a plurality of detectors, a plurality of photon number distributions can be obtained by one-time illumination, which is equivalent to indirectly realizing the relative movement of the illumination spot position to the fluorescent molecule, and theoretically, the speed can be increased by 4 times compared with the traditional two-dimensional dark spot positioning method.
Further preferably, the detector array has seven single detectors.
Further preferably, according to the number of photons received by different monomer detectors and the parameters of the hollow optical plate irradiating the fluorescent molecule sample, the maximum likelihood probability for generating the photon flow distribution can be obtained, and the luminous position of the fluorescent molecule can be obtained by inverse solution.
Preferably, the excitation light and the loss light are projected to the sample through collimation, phase modulation and polarization characteristic change in sequence to form a hollow light spot.
Preferably, the laser pulses of the excitation light and the loss light coincide in time.
The principle of the invention is as follows:
the two-dimensional dark spots used in the past can only reach a diameter of about 200nm at the diffraction limit, so that the two-dimensional dark spots can only be positioned under the condition of sparse fluorescent molecule distribution. The idea of super-diffractive dark spots is derived from Stimulated Emission Depletion Microscopy (STED), with the difference that the mode of excitation light is changed from a solid spot to a hollow spot. By utilizing the stimulated emission loss principle, one beam of hollow spot is used for excitation, and the other beam of hollow spot loses fluorescence, the super-diffraction dark spot can be obtained, and the size of the super-diffraction dark spot can be reduced to about 50 nm. Theoretically, under the condition that two-dimensional space obtains equal positioning accuracy, the concentration of the dye realized by the invention can be 16 times of that of the existing experimental method, and the dye concentration can better meet the requirements of real biological environment.
For the optical signals received by the detector array, the invention adopts the maximum likelihood probability estimation function and carries out inverse solution, thus obtaining more accurate fluorescent molecule positioning. Under the condition of adding noise, the positioning precision can still reach sub-ten nanometers, and is improved compared with the existing method.
The maximum likelihood function estimation formula is as follows:
wherein n isiRepresenting the number of photons received at the i-th detector, piRepresents the ratio of the relative illumination intensities of the different detectors in their conjugate planes, i.e.:
the concentration of dye solution of the existing MINFUX localization method is limited to relatively sparse situation, but the reduced super-diffraction dark spot can locate more dense dye molecules. And based on a parallel detection mechanism, the positioning speed can be greatly improved.
Corresponding to the method, the invention also provides a two-dimensional sub-ten nanometer positioning device based on parallel detection, which comprises a light source, a sample stage for bearing a sample to be detected and a parallel detection system for collecting signal light emitted by the sample; the light source comprises a first light source emitting exciting light and a second light source emitting loss light;
a 0-2 pi vortex phase plate for light beam phase modulation is arranged on the light path of the first light source and the second light source, so that emitted exciting light and lost light are converged into a hollow light spot to illuminate a sample and a fluorescent molecule is excited to emit signal light;
the parallel detection system collects signal light for acquiring images at corresponding scanning positions and position information of fluorescent molecules.
Preferably, a single-mode fiber and a collimating lens for filtering the laser beam are sequentially arranged between the first light source and the second light source and the 0-2 pi vortex phase plate.
Preferably, the 0-2 pi vortex phase plate has a variable modulation function
Where ρ is the distance between a point on the beam and the optical axis,
is the included angle between the position polar coordinate vector in the section plane of the light beam vertical to the optical axis and the x axis.
Preferably, the parallel detection system has a detector array comprising a plurality of single detectors, each receiving a different proportion of the number of photons.
In the invention, the hollow light spots of the excitation light and the loss light are overlapped at the center of the light spot in space; two laser pulses emitted by the first light source and the second light source are overlapped in time by utilizing a time delay circuit connected with the first light source and the second light source.
Therefore, compared with the existing MINLUX method, the method has the following beneficial technical effects:
(1) the two-dimensional sub-ten nanometer super-resolution microscopic positioning method based on the detector array is provided for the first time;
(2) localization can be performed in an environment with a higher concentration of dye molecules than with MINLUX;
(3) the device is simple and convenient to operate.
Drawings
FIG. 1 is a schematic structural view of a super-resolution microscopy apparatus according to the present embodiment;
FIG. 2 is a normalized light intensity distribution curve of the excited hollow spot according to the present embodiment;
FIG. 3 is a normalized light intensity distribution curve of the lossy hollow spot of this embodiment;
FIG. 4 is a normalized light intensity distribution curve of the super-diffractive dark spot of the present embodiment;
FIG. 5 is a simulation diagram of the position distribution of the parallel detection conjugate plane according to the present embodiment;
fig. 6 is a simulation diagram of positioning accuracy in this embodiment.
Detailed Description
The present invention will be described in detail with reference to the following examples and drawings, but the present invention is not limited thereto.
As shown in fig. 1, the super-resolution microscopic positioning apparatus of the present embodiment includes: the optical fiber scanning device comprises a first laser 1, a second laser 8, a first single-mode fiber 2, a second single-mode fiber 9, a first collimating lens 3, a second collimating lens 10, a first 0-2 pi vortex phase plate 4, a second 0-2 pi vortex phase plate 11, a first 1/2 wave plate 5, a second 1/2 wave plate 12, a first 1/4 wave plate 6, a second 1/4 wave plate 13, a first reflecting mirror 7, a second reflecting mirror 15, a third reflecting mirror 19, a fourth reflecting mirror 24, a first dichroic mirror 14, a second dichroic mirror 16, an achromatic 1/4 wave plate 17, a scanning vibrating mirror 18, a scanning lens 20, a field mirror 21, a microscope objective lens 22, a sample stage 23, a band-pass filter 25, a microlens array 26, a pinhole array 27, an optical fiber bundle 28, a detector array 29 and a delay circuit 30.
The first single-mode fiber 2, the first collimating lens 3, the first 0-2 pi vortex phase plate 4, the first 1/2 wave plate 5 and the first 1/4 wave plate 6 are sequentially positioned on an optical axis of an emergent light beam of the first laser 1; the second single-mode fiber 9, the second collimating lens 10, the second 0-2 pi vortex phase plate 11, the second 1/2 wave plate 12 and the second 1/4 wave plate 13 are sequentially located on an optical axis of an emergent light beam of the second laser 8.
The first dichroic mirror 14 is located on the optical axis of the light beam modulated by the first 0-2 pi vortex phase plate 4 and the second 0-2 pi vortex phase plate 11. The second dichroic mirror 16 and the achromatic 1/4 wave plate 17 are sequentially located on the optical axis of the emergent light of the first dichroic mirror 14 after being deflected by the second reflecting mirror 15; the scanning galvanometer 18 is positioned on the optical axis of the light beam after exiting through the second dichroic mirror 16.
The scanning lens 20, the field lens 21, the microscope objective 22 and the sample stage 23 are sequentially positioned on the optical axis of the emergent beam of the scanning galvanometer 18.
The band-pass filter 25, the micro-lens array 26, the pinhole array 27, the optical fiber bundle 28 and the detector array 29 are sequentially positioned on the optical axis of the light beam reflected by the reflector after passing through the second dichroic mirror 16; the pinhole array 27 is located at the focal plane of the microlens array 26.
In this embodiment, the first laser 1 is connected to the second laser 8 by a delay circuit 30, which is used to adjust the coincidence of the two laser pulses in time.
In this embodiment, the numerical aperture NA of the microscope objective lens 22 is 1.4; the pinhole 27 used has a diameter of 0.2 airy disk and the detector array 29 is an avalanche diode Array (APD).
The method for performing super-resolution microscopic positioning by using the device shown in FIG. 1 is as follows:
laser beams emitted from the first laser 1 and the second laser 8 are subjected to pulse coincidence in time through the delay circuit 30, are guided into the first single-mode fiber 2 and the second single-mode fiber 9, and are collimated through the first collimating lens 3 and the second collimating lens 10. The collimated light beams are incident to the first 0-2 pi vortex phase plate 4 and the second 0-2 pi vortex phase plate 11 to be modulated, and hollow light spots are generated.
The two beams of light respectively emitted by the first 0-2 pi vortex phase plate 4 and the second 0-2 pi vortex phase plate 11 are changed into linearly polarized light through the first 1/2 wave plate 5 and the second 1/2 wave plate 12, and then changed into right-handed circularly polarized light through the first 1/4 wave plate 6 and the second 1/4 wave plate 13. Recombined into a beam of light by the first dichroic mirror 14. Then, the polarization state change caused by the dichroic mirror is compensated by the achromatic 1/4 wave plate 17, and the polarization state is changed to right-handed circularly polarized light again.
The modulated light is incident on the scanning galvanometer 18, and the light beam emitted by the scanning galvanometer 18 is focused by the scanning lens 20, collimated by the field lens 21, and projected onto a sample to be measured on the sample stage 23 through the microscope objective 22.
The optical field distribution of the incident light near the focal point of the microscope objective 22 can be determined by debye integration, as follows:
in the formula (I), the compound is shown in the specification,
is a coordinate in a cylindrical coordinate system with the focal position of the microscope objective lens 22 as the origin,
represent
The intensity of electric vector is represented by i as an imaginary unit, C as a normalization constant, theta is a beam aperture angle, phi is an included angle between a position polar coordinate vector and an x axis in a cross section of the beam vertical to the Z axis, and A1(θ, φ) is the amplitude distribution of the incident light, A2(theta, phi) characterizes the structure of the microscope objective lens 22,
then the polarization information of the incident light is represented, k is 2 pi/lambda, and n is the medium refractive index.
It can be found from the above formula calculation that the light spot formed on the sample to be measured after the two incident circularly polarized lights are focused by the microscope objective lens 22 is a doughnut-shaped hollow light spot, and the specific light field distribution normalization curves of the two light spots are respectively shown in fig. 2 and 3.
The signal light emitted by the sample to be measured is collected by the microscope objective 22, then passes through the field lens 21, the scanning lens 20 and the scanning galvanometer 18 in sequence, passes through the second dichroic mirror 16, and is finally reflected. The signal beam is filtered from stray light by a bandpass filter 25, focused by a microlens array 26 and spatially filtered by a pinhole array 27 and finally received by a detector array 29.
The controller adjusts the scanning galvanometer 18 to realize two-dimensional scanning of the sample to be detected.
By using stimulated emission depletion principle, one beam of hollow spot is used for excitation, and the other beam of hollow spot loses fluorescence, the super-diffraction dark spot can be obtained, and the size of the super-diffraction dark spot can be reduced to about 50nm, as shown in figure 4.
With the detector array, seven detectors can obtain seven photon number distributions under one illumination. By using the maximum likelihood probability estimation function and performing inverse solution, a more accurate fluorescent molecule location can be obtained, for example, fig. 5 shows the equivalent illumination distribution of seven detectors on a conjugate plane under one illumination condition. With the addition of noise, the positioning accuracy can still reach sub-ten nanometers, as shown in fig. 6.
In this embodiment, although not shown in fig. 1, the apparatus in this embodiment should further include a computer connected to each detector, and process the optical signals received by the detector array to obtain the image at the corresponding scanning position and the position information of the fluorescent molecules.
The above description is only exemplary of the preferred embodiments of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A two-dimensional sub-ten nanometer positioning method based on parallel detection is characterized by comprising the following steps:
1) the exciting light is converged into a hollow light spot to illuminate the sample, so that the sample is in an exciting state;
2) the lost light is converged into a hollow light spot illumination sample, and the hollow light spot illumination sample is superposed with the center of the hollow light spot of the exciting light to excite the fluorescent molecules to emit signal light;
3) and collecting the signal light, and acquiring an image at a corresponding scanning position and position information of the fluorescent molecules through the received signal light.
2. The two-dimensional sub-ten-nanometer positioning method based on parallel detection according to claim 1, wherein in step 3), the signal light is received by a detector array comprising a plurality of single detectors, each of which receives a different proportion of photon numbers.
3. The two-dimensional sub-ten nanometer positioning method based on parallel detection as claimed in claim 2, wherein according to the number of photons received by different single detectors and the parameters of the hollow optical plate illuminating the fluorescent molecule sample, the maximum likelihood probability for generating the photon flow distribution can be obtained, and the light emitting position of the fluorescent molecule can be obtained by inverse solution.
4. The two-dimensional sub-ten nanometer positioning method based on parallel detection as claimed in claim 1, wherein the excitation light and the loss light are projected to the sample sequentially through collimation, phase modulation and polarization change to form a hollow light spot.
5. The parallel-detection-based two-dimensional sub-ten-nanometer positioning method of claim 1, wherein the laser pulses of the excitation light and the loss light are temporally coincident.
6. A two-dimensional sub-ten nanometer positioning device based on parallel detection comprises a light source, a sample stage for bearing a sample to be detected and a parallel detection system for collecting signal light emitted by the sample; the method is characterized in that: the light source comprises a first light source emitting exciting light and a second light source emitting loss light;
a 0-2 pi vortex phase plate for light beam phase modulation is arranged on the light path of the first light source and the second light source, so that emitted exciting light and lost light are converged into a hollow light spot to illuminate a sample and a fluorescent molecule is excited to emit signal light;
the parallel detection system collects signal light for acquiring images at corresponding scanning positions and position information of fluorescent molecules.
7. The parallel-probing-based two-dimensional sub-ten-nanometer positioning apparatus of claim 6, wherein: and a single-mode fiber and a collimating lens for filtering the laser beam are sequentially arranged between the first light source and the second light source and between the 0-2 pi vortex phase plate.
8. The parallel-probing-based two-dimensional sub-ten-nanometer positioning apparatus of claim 6, wherein: the 0-2 pi vortex phase plate has a variable modulation function
Where ρ is the distance between a point on the beam and the optical axis,
is the included angle between the position polar coordinate vector in the section plane of the light beam vertical to the optical axis and the x axis.
9. The parallel-probing-based two-dimensional sub-ten-nanometer positioning apparatus of claim 6, wherein: the parallel detection system has a detector array comprising a plurality of individual detectors, each individual detector receiving a different proportion of photon counts.
10. The parallel-probing-based two-dimensional sub-ten-nanometer positioning apparatus of claim 6, wherein: the hollow light spots of the excitation light and the loss light are overlapped at the center of the light spot in space;
two laser pulses emitted by the first light source and the second light source are overlapped in time by utilizing a time delay circuit connected with the first light source and the second light source.
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