CN113532271B - Mark-free three-dimensional super-resolution microscopy method and device - Google Patents
Mark-free three-dimensional super-resolution microscopy method and device Download PDFInfo
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Abstract
The invention discloses a label-free three-dimensional super-resolution microscopic method, which comprises the following steps: carrying out two-dimensional scanning on the unmarked sample by using the solid illumination light beam and the hollow illumination light beam simultaneously to obtain a positive confocal reflected light intensity graph obtained by modulating the solid light spots and a negative confocal reflected light intensity graph obtained by modulating the hollow light spots, and carrying out differential calculation; the axial super-resolution is realized by scanning and measuring the sample by adopting a differential measurement scanning method, and a photoelectric detector is arranged at the defocusing distance-u of a focal plane M At the off-focus distance + u of the other photodetector at the focal plane M Respectively measuring intensity curves reflecting the surface appearance change of the sample, carrying out differential subtraction and normalization processing to obtain a differential intensity signal; reconstructing the surface appearance and the microscale of the sample according to the light intensity of the intensity curve in the AB linear measurement range near the zero point; and obtaining a three-dimensional super-resolution microscopic image.
Description
Technical Field
The invention belongs to the field of laser point scanning microscopy, and particularly relates to a non-marking three-dimensional super-resolution microscopy method and a non-marking three-dimensional super-resolution microscopy device.
Background
The label-free super-resolution microscope is mainly suitable for label-free non-fluorescent samples. To realize a label-free super-resolution microscope, a simple method is a near-field scanning optical microscope (SNOM) technique, which detects a near-field signal of a sample surface using an ultra-thin nanotip. However, the low imaging speed and the requirement for close control of the distance between the tip and the object surface make SNOM less than satisfactory. In subsequent developments, scientists have used some nanostructures to achieve multiple label-free super-resolution microscopy, such as superlens microspheres with hyperbolic profile, solid immersion lens based metamaterials (mSIL), and nanowire annular illumination microscopy (NWRIM), among others. However, the above techniques also have certain application bottlenecks. On the one hand, nanostructure-based devices must be placed in the near field of the object and are limited to surface detection only. On the other hand, these techniques impose complex requirements on the manufacturing process and also increase the costs.
The technology of the unmarked far-field optical microscope is used for measuring the surface topography of the three-dimensional object, the structure of the technology is simpler than that of SNOM, and the technology is mainly characterized by utilizing the interference property of light, such as white light interferometer. The technology utilizes the coherent enhancement of light with different wavelengths in different optical path differences, and the color corresponding to the wavelength of the coherent enhancement reflects the optical path information of the point of the object and also the depth information of the object. Although a white light interferometer can achieve high axial resolution, its lateral resolution is not high and is limited by diffraction limit.
Confocal microscopy is a technique that can simultaneously improve axial resolution and axial resolution, while the improved intensity differential technique on confocal systems can further improve lateral and axial resolution. Intensity differentiation techniques have the specific characteristics of being easy to implement and not limited to a sample, and improve the spatial resolution of the microscope in a more economical manner. It was proposed in the 90 s of the 20 th century that subtracting confocal images from wide-field images could improve resolution while reflecting object depth information. However, this method only weakens the low frequency information on the frequency spectrum, amplifies the high frequency information, and thus reduces the signal-to-noise ratio of the image, and therefore, it is not well applied.
The Differential Confocal Microscopy (DCM), described in patent application publication No. CN102830102A, successfully improved axial resolution by differential subtraction using a bi-confocal setup. The nano-scale axial detection is realized, which is beneficial to the fluctuation detection of the surface topography of the material.
The fluorescence differential microscopy (FED) described in patent application publication No. CN110220875A excites a sample with two different excitation lights, a solid spot and a hollow spot, and subtracts the image of the hollow spot from the image of the solid spot, and since this subtraction process is equivalent to reducing the point spread function, lateral super-resolution is achieved. In subsequent improvement, a parallel scanning device is added, so that the scanning time is reduced, and real-time imaging is realized. The transverse resolution can be effectively improved by applying the FED method to the unmarked microscopy.
Disclosure of Invention
On the basis of laser point scanning microscopy, the invention combines the respective characteristics of transverse parallel radiation differential super-resolution technology and differential confocal microscopy, provides a transverse optical super-resolution and axial nano-resolution three-dimensional super-resolution imaging system, and realizes the surface morphology detection of unmarked non-fluorescent samples and materials.
The axial resolution of the imaging system is improved by a differential confocal microscopic detection technology, and the transverse resolution is improved by a parallel radiation differential super-resolution method.
The specific technical scheme of the invention comprises the following steps:
(1) the method comprises the steps of simultaneously carrying out two-dimensional scanning on a non-marked sample by utilizing a solid illumination light beam and a hollow illumination light beam to obtain an orthophoto-confocal reflected light intensity diagram I obtained by modulating a solid light spot s (x,y,z 0 0) and a negative confocal reflected light intensity map I obtained by modulation of the hollow light spot k (x,y,z 0 ,0)；
(2) According to formula I f (x,y,z 0 ,0)＝I s (x,y,z 0 ,0)-αI k (x,y,z 0 0) obtaining a transverse super-resolution image I f (x,y,z 0 0), wherein alpha is a weight factor which can be determined according to the actual imaging effect, and in the calculation process, a direct zeroing mode is adopted possibly because the difference has a negative intensity problem;
(3) the axial super-resolution is realized by scanning and measuring the sample by adopting a differential measurement scanning method, and a photoelectric detector is arranged at the defocusing distance-u of a focal plane M At the off-focus distance + u of the other photodetector at the focal plane M Respectively measuring an intensity curve I reflecting the surface appearance change of the sample A (x,y,z,-u M ) And I B (x,y,z,+u M ) Performing a differential subtraction and normalization processThen obtaining a differential intensity signal I D (x,y,z,u M )；
(4) Optimization of u M Value, increase of axial resolution of differential confocal method, then according to intensity curve I D (x,y,z,u M ) And (5) reconstructing the surface appearance and the microscale of the sample according to the light intensity in the AB linear measurement range near the zero point.
(5) And obtaining a three-dimensional super-resolution microscopic image by combining the transverse super-resolution image information and the axial differential measurement information.
The principle of the invention is as follows:
assuming (x, y, z) is the spatial coordinate of the sample, the image raw data obtained by this method can be denoted as I (x, y, z, u) M ) Wherein u is M Is the defocus distance of the detector.
For convenient calculation and analysis, coordinate transformation is carried out on each variable:
radial optical coordinate v:
a is the pupil radius, and f is the focal length of the objective lens; radial coordinate in polar coordinates
Axial optical coordinate u:
normalized radius
J 0 Is a zero order bessel function.
The pupil function of the microscope objective in the device is P (ρ), and according to the confocal theory, the three-dimensional point spread function is expressed as:
(1) implementation of lateral super-resolution
Using solid excitation spots and transverse hollow excitation spotsAnd scanning light spots in two excitation modes to respectively obtain two images. Both excitation mode detectors are in the focal plane, so u M 0. Pupil function P with solid excitation s (rho) is 1, | rho | is less than or equal to 1, the obtained common confocal image is obtained, and the intensity distribution is recorded as I s (x, y, z, 0). When the transverse hollow excitation is carried out, the excitation light passes through the vortex phase plate, and the pupil function becomes
The resulting negative confocal image had an intensity distribution denoted I k (x,y,z,0)。
Transverse super-resolution image I f (x, y, z,0) is the result of a special weight difference between the confocal image and the negative confocal image, and the expression is:
I f (x,y,z,0)＝I s (x,y,z,0)-αI k (x,y,z,0) (2)
in the formula I s (x, y, z,0) and I k The (x, y, z,0) is normalized intensity distribution during calculation, and alpha is a weight factor and can be determined according to the actual imaging effect. In the calculation process, it is possible that the difference has a negative strength problem, and a direct zeroing mode is generally adopted.
(2) Implementation of longitudinal super-resolution
For analyzing the longitudinal resolution in the system, the detected object is an ideal point, the detector is located in the focal plane, and only the light intensity distribution axially distributed at the center point is considered, i.e. x is 0, y is 0, upsilon is 0, and u is 0 in formula (2) M ＝0：
Its function curve is shown in the short-dashed line I of FIG. 3 0 。
As can be seen from this diagram, when the axial position u changes around u-0, the light intensity I is present 0 The variation is not large, which means that if the scan plane of the sample is slightly off the focal plane, the intensity variation is not significant. This means that the maximum light intensity conventionally obtained by confocal system detection is obtainedAnd thus the resolution of the method of detecting changes in the height of the object surface is not high.
And the light intensity I is far away from u-0 0 The change of the axial position u is in a more obvious linear relation. Such a significant intensity change may more significantly reflect the height change of the object surface.
By arranging two detection light paths and deviating the detectors to the same distance + u in opposite directions M And-u M . The two paths of detected light intensity I A ,I B The function curve with axial position u is:
I A see solid line, I, of FIG. 3 B See the long dashed line of fig. 3.
The two paths of detection signals are subtracted to obtain a differential signal I D ：
Differential signal I D See the chain line of fig. 3, from which it can be seen that the curve I is obtained by subtracting the two signals D Having a linear region AB of greater slope (relative to the original curve I without subtraction) A Linear region CD) in (1), which means equally spaced light intensities I D The axial position u varies more significantly with the variation, which means a higher axial resolution.
(3) Realization of three-dimensional super-resolution
Scanning the object by galvanometers at a certain axial position z 0 Performing two-dimensional scanning, axially scanning the object by axially moving the objective lens, and performing one-time two-dimensional scanning to obtain four groups of data I s (x,y,z 0 ,0),I k (x,y,z 0 ,0),I A (x,y,z 0 ,-u M ),I B (x,y,z 0 ,+u M )。
According to the principle of realizing the transverse and axial super resolution, a vortex phase plate is arranged in one excitation light path, and detectors are respectively arranged at focal plane positions and deviated from the focal plane + u in a plurality of detection light paths M And-u M Location. Finally, subtracting the detected signals according to a corresponding principle to obtain transverse super-resolution information:
I f (x,y,z 0 ,0)＝I s (x,y,z 0 ,0)-αI k (x,y,z 0 ,0) (7)
differential signals I obtained from different scanning points (x, y) D ：
I D (x,y,z 0 ,u M )＝I A (x,y,z 0 ,-u M )-I B (x,y,z 0 ,+u M ) (8)
The axial information of the object needs to be embodied by the change of the height h of the object. By obtaining differential signals I of points in the transverse direction D (x,y,z 0 ,u M ) Value of (d), find z and differential signal I D Obtaining the axial position information z (relative to the scanning plane z) of each point (x, y) in the transverse direction of the object by corresponding conversion relation 0 ) And converted into object height information h.
Drawings
FIG. 1 is a schematic view of a first embodiment of a microscope arrangement according to the invention;
FIG. 2 is a schematic view of a second embodiment of the microscope apparatus according to the present invention;
FIG. 3 is a schematic diagram of the microscope apparatus according to the present invention for realizing the principle of axial super resolution.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below. Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1
As shown in fig. 1, a non-marking differential three-dimensional super-resolution microscopic device comprises a laser 1, a lens 2, a polarizer 3, a vortex phase plate 4, a half-wave plate 5, a polarization beam splitter 6, a quarter-wave plate 7, a dichroic mirror 8, a scanning mirror 9, a scanning mirror 10, a scanning lens 11, a tube lens 12, a reflecting mirror 13, a microscope objective 14, a sample 15, a lens 16, an aperture 17, a photodetector 18, a laser 19, a lens 20, a polarization beam splitter 21, a quarter-wave plate 22, a beam splitter 23, a lens 24, an aperture 25, a photodetector 26, a beam splitter 27, a lens 28, an aperture 29, a photodetector 30, a lens 31, an aperture 32, a photodetector 33 and a computer 34.
The unmarked differential three-dimensional super-resolution microscopy method realized by adopting the device shown in FIG. 1 comprises the following steps:
(1) the laser 1 emits illumination light, the illumination light is collimated by the lens 2, is adjusted into linearly polarized light by the polarizer 3, is modulated into a hollow light spot by the vortex phase plate 4, the incident light is modulated into p-polarized light by the half-wave plate 5, and the linearly polarized light is adjusted into circularly polarized light by the quarter-wave plate 7;
(2) the laser 19 emits illumination light, the illumination light is collimated by the lens 20, and emergent light passing through the polarization beam splitter 21 is adjusted to be circularly polarized by the quarter-wave plate 22;
(3) lasers 1 and 19 emit laser light with wavelengths close to but different from each other, preferably, the central wavelengths are 405nm and 450nm respectively, the dichroic mirror 8 can transmit light (405nm) with shorter wavelength emitted by the laser 1 and reflect light (450nm) with longer wavelength emitted by the laser 19, and the two laser beams enter the mirror vibrating module after passing through the dichroic mirror 8;
(4) the light emitted by the dichroic mirror 8 reaches a scanning galvanometer 9 and a scanning galvanometer 10, and a galvanometer module consisting of the scanning galvanometer 9 and the scanning galvanometer 10 is controlled by a computer 34 so as to perform two-dimensional scanning on the sample;
(5) two paths of laser pass through a scanning lens 11 and a tube lens 12, are reflected by a reflector 13, enter a microscope objective 14 and illuminate a sample 15;
(6) the two-color reflected light of the sample is collected by the same objective lens 14, returns to the galvanometer module and is divided into two paths to the dichroic mirror 8 according to the wavelength;
(7) the reflected light with shorter wavelength (405nm) penetrates through the dichroic mirror 8, the s component is reflected by the polarization beam splitter 6, converged by the lens 16 and the small hole 17, and finally collected by the photoelectric detector 18 positioned at the focal position to obtain a hollow light spot scanning image I k (x,y,z 0 ,0)；
(8) The reflected light (450nm) with longer wavelength is reflected by the dichroic mirror 8, passes through the quarter wave plate 22, the s component is reflected by the polarization beam splitter 21, is split into two beams by the beam splitter 23, one beam passes through the lens 24 and the small hole 25 and is collected by the photoelectric detector 26 positioned at the focus position, and the solid light spot scanning image I is obtained s (x,y,z 0 0), the other beam is split into two beams by the beam splitter 27, and the two beams are respectively focused by the lenses 28 and 31 with the same focal length;
(9) scanning solid spots in image I s (x,y,z 0 0) and hollow spot scan image I k (x,y,z 0 0) carrying out special weight subtraction after normalization to obtain a transverse super-resolution graph I f (x,y,z 0 ,0)；
(10) Photodetector 30 is positioned at defocus distance-u from the focal plane of lens 28 M At, the photodetector 33 is positioned at the defocus distance + u of the focal plane of the lens 31 M Respectively measuring an intensity curve I reflecting the surface topography change of the sample 15 A (x,y,z,-u M ) And I B (x,y,z,+u M ) Carrying out differential subtraction and normalization processing to obtain a differential intensity signal I D (x,y,z,u M )；
(11) Optimization of u M Value, increase of axial resolution of differential confocal method, then according to intensity curve I D (x,y,z,u M ) And reconstructing the surface appearance and the microscale of the sample according to the light intensity in the AB measurement range near the zero point.
Example 2
As shown in fig. 2, a fluorescence radiation differential microscopy device for realizing three-dimensional super-resolution of a sample comprises a laser 1, a lens 2, a half-wave plate 3, a polarization beam splitter 4, a vortex phase plate 5, a polarization beam splitter 6, reflecting mirrors 7 and 8, a quarter-wave plate 9, a half-wave plate 10, a polarization beam splitter 11, a quarter-wave plate 12, a dichroic mirror 13, a scanning mirror 14, a scanning mirror 15, a scanning lens 16, a tube lens 17, a reflecting mirror 18, a microscope objective lens 19, a sample 20, a lens 21, an aperture 22, a fiber-coupled input photodetector 23, a fiber-coupled input photodetector 24, a laser 25, a lens 26, a polarization beam splitter 27, a quarter-wave plate 28, a beam splitter 29, a lens 30, an aperture 31, a photodetector 32, a lens 33, an aperture 34, a photodetector 35 and a computer 36.
(1) The laser 1 emits illumination light, the illumination light is collimated by the lens 2, the half-wave plate 3 adjusts the light intensity ratio of p-polarized light and s-polarized light passing through the polarization beam splitter 4, the p-polarized light is modulated into a hollow light beam by the vortex phase plate 5, the s-polarized light (solid light beam) passes through the reflectors 7 and 8, and the polarization beam splitter 6 combines the hollow light beam and the solid light beam, wherein the two light beams have a small distance difference d;
(2) according to the formula of diffraction limit of circular hole
Where k is the wave vector, a is the radius of the circular hole, θ is the aperture angle, I 0 The central light intensity maximum value, the diameter of the Airy spot can be calculated by the formula
A secondary large relative intensity of I is obtained 2 ≈0.00175I 0 Therefore, when the distance between two light spots is greater than one Airy spot distance, the influence of the side lobe is less than two thousandth, and therefore the distance between the two light spots is greater than one Airy spot to ensure that the light spots are not influenced mutually. In the embodiment, in order to ensure that the solid light spots and the hollow light spots cannot interfere with each other, the solid light spots and the hollow light spots are staggered on the object plane by at least more than 200nm by utilizing the adjusting reflectors 7 and 8;
(3) the two beams of light are adjusted into circularly polarized light through a quarter-wave plate 9 and a half-wave plate 10, wherein p components of the two beams of light penetrate through a polarization beam splitter 11 and are adjusted into circularly polarized light through a quarter-wave plate 12;
(4) the laser 25 emits illumination light, the illumination light is collimated by the lens 26, and emergent light passing through the polarization beam splitter 27 is adjusted to be circularly polarized by the quarter-wave plate 28;
(5) lasers 1 and 25 emit laser light with wavelengths close to but different from each other, preferably, the central wavelengths are 405nm and 450nm respectively, the dichroic mirror 13 can transmit light (405nm) with shorter wavelength emitted by the laser 1 and reflect light (450nm) with longer wavelength emitted by the laser 25, and the two laser beams enter the mirror-vibrating module after passing through the dichroic mirror 13;
(6) the light emitted by the dichroic mirror 13 reaches the scanning galvanometer 14 and the scanning galvanometer 15, and a galvanometer module formed by the scanning galvanometer 14 and the scanning galvanometer 15 is controlled by a computer 36 so as to perform two-dimensional scanning on the sample;
(7) two paths of laser pass through a scanning lens 16 and a tube lens 17, are reflected by a reflector 18, enter a microscope objective 19 and illuminate a sample 20;
(8) the two-color reflected light of the sample is collected by the same objective lens 19, returns to the galvanometer module and is divided into two paths according to the wavelength at the dichroic mirror 13;
(9) the reflected light (405nm) with shorter wavelength penetrates through the dichroic mirror 13, passes through the quarter-wave plate 12, the s component is reflected by the polarization beam splitter 11, is converged by the lens 21, the two reflected light beams with small displacement difference are respectively received by the two optical fibers 22 and are finally collected by the photoelectric detectors 23 and 24, and the on-focus solid spot scanning image I is obtained s (x,y,z 0 0) and hollow spot scanning image I k (x,y,z 0 0); the two fluorescence intensity maps are subjected to shift matching according to formula I f (x,y,z 0 ,0)＝I s (x,y,z 0 ,0)-αI k (x,y,z 0 0) obtaining a super-resolution image I f (x,y,z 0 ,0)；
(10) The reflected light with longer wavelength (450nm) is reflected by the dichroic mirror 8, wherein the s component is reflected by the polarizing beam splitter 27, is split into two beams by the beam splitter 23, one beam passes through the lens 24 and the small hole 25 and is collected by the photoelectric detector 26, and the on-focus solid light spot scanning image I is obtained s (x, y, z,0), and the other beam is split into two beams by the beam splitter 27Are focused by lenses 28 and 31, respectively, of the same focal length;
(11) photodetector 32 is positioned at defocus distance-u from the focal plane of lens 30 M At the focal distance + u, the photodetector 35 is disposed at the focal plane of the lens 33 M Respectively measuring an intensity curve I reflecting the surface topography change of the sample 20 A (x,y,z,-u M ) And I B (x,y,z,+u M ) Carrying out differential subtraction and normalization processing to obtain a differential intensity signal I D (x,y,z,u M )；
(12) Optimization of u M Value, increase of axial resolution of differential confocal method, then according to intensity curve I D (x,y,z,u M ) And reconstructing the surface appearance and the microscale of the sample according to the light intensity in the AB measurement range near the zero point.
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 (6)
1. A label-free three-dimensional super-resolution microscopy method is characterized by comprising the following steps:
(1) the solid illumination light beam and the hollow illumination light beam are used for simultaneously carrying out two-dimensional scanning on the unmarked sample to obtain an orthophoto confocal reflected light intensity diagram obtained by modulating the solid light spots
And a negative confocal reflected light intensity map obtained by modulating the hollow light spot
；
(2) According to the formula
Obtaining a transverse super-resolution image
WhereinαIn order to be a weight factor, the weight factor,
(3) the axial super-resolution is realized by scanning and measuring the sample by adopting a differential measurement scanning method, and a photoelectric detector is arranged in the defocusing distance of a focal plane
At the off-focus distance of the focal plane
Respectively measuring intensity curves reflecting the surface appearance change of the sample
And
carrying out differential subtraction and normalization processing to obtain a differential intensity signal
；
(4) Optimization
Value according to intensity curve
Reconstructing the surface appearance and the microscale of the sample according to the light intensity in the AB linear measurement range near the zero point;
(5) obtaining a three-dimensional super-resolution microscopic image by combining transverse super-resolution image information and axial differential measurement information;
two lasers with similar but different wavelengths are used for emitting laser, and the solid illumination light beam and the hollow illumination light beam are respectively formed; one path of laser has the wavelength of 405 nm; the other laser wavelength is 450 nm.
2. The markerless three-dimensional super resolution microscopy method according to claim 1, wherein in steps (1) and (3) the reflected laser intensity signal is collected using an Avalanche Photodiode (APD) or a photomultiplier tube (PMT).
3. The method according to claim 1, wherein the calculation result is directly zeroed when the formula difference in step (2) has negative values.
4. The label-free three-dimensional super-resolution microscopy method according to claim 1,
and
the normalized intensity distribution is taken for the calculation,αis a weighting factor.
5. The markerless three-dimensional super-resolution microscopy method according to claim 1, wherein two detection light paths are arranged and the detectors are respectively deviated in opposite directions by the same distance
And
。
6. an unmarked three-dimensional super-resolution microscopy imaging device based on the unmarked three-dimensional super-resolution microscopy method as claimed in any one of claims 1 to 5, characterized in that two lasers emit laser light with similar but different wavelengths, and the dichroic mirror transmits the light with shorter wavelength emitted by one laser and reflects the light emitted by the other laserFor light with longer wavelength, two paths of laser enter a galvanometer module to scan a sample simultaneously after passing through a dichroic mirror; wherein the laser scanning is carried out at 405nm
Obtained by scanning with a 450nm laser
、
、
Two paths of signals are separated through different wavelengths.
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