CN113466090A - Surface plasmon imaging system based on difference denoising - Google Patents
Surface plasmon imaging system based on difference denoising Download PDFInfo
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- CN113466090A CN113466090A CN202110719599.4A CN202110719599A CN113466090A CN 113466090 A CN113466090 A CN 113466090A CN 202110719599 A CN202110719599 A CN 202110719599A CN 113466090 A CN113466090 A CN 113466090A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
- G01N15/0211—Investigating a scatter or diffraction pattern
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
- G01N15/0227—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging using imaging, e.g. a projected image of suspension; using holography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N2015/0038—Investigating nanoparticles
Abstract
The invention discloses a surface plasmon imaging system based on differential denoising, which comprises: the device comprises a rotary illumination system, a tube lens, a beam splitter, an oil immersion microscope objective, a gold film substrate, a high-precision displacement platform and an imaging light path; the surface plasmon has the characteristics of local field enhancement and sensitivity to environmental refractive index change, and can be used for imaging tiny particles. Firstly, the rotating illumination system is utilized to realize that incident light is used for rotating illumination of an object, interference fringes generated by the incident light and scattered light are eliminated, and an image without the interference fringes and with a small size is obtained. On the basis, a differential denoising method is utilized, the high-precision displacement platform is rapidly moved, background noise of the image is removed, and the signal-to-noise ratio of the image is improved. The imaging system greatly improves the signal-to-noise ratio of surface plasmon microscopic imaging by using a differential denoising method, can perform high-contrast imaging on nanoparticles with the diameter as low as 22nm, and has the particle size resolution of 10 nm.
Description
Technical Field
The invention relates to the field of surface optical microscopic imaging with high signal-to-noise ratio, in particular to surface plasmon imaging based on differential denoising.
Background
Optical detection methods of single nanoparticle particle size have been widely used in biophysical studies and are expanding to applications including national security, environmental monitoring, and early diagnosis. The technology provides an important means for exploring the basic characteristics of biological and photonic materials and thoroughly changes the understanding of the biophysical phenomena occurring on the nanometer scale. Nanoparticles with smaller diameters tend to be more harmful to human health than larger nanoparticles, as they can penetrate into the lung and can then induce lung cancer, respiratory diseases and even organ dysfunction. Therefore, we should focus more on the detection of ultra-fine particles, especially nanoparticles with diameters less than 100 nm.
Surface acoustic waves are electromagnetic modes localized in the near-field region of the Surface of an object, and Surface plasmons (SPPs) present at the interface between metal and dielectric are common Surface acoustic waves. When electromagnetic waves are incident to the interface between metal (usually noble metal, such as gold or silver) and dielectric, free electrons on the surface of the metal oscillate collectively, and the electromagnetic waves are coupled with the free electrons on the surface of the metal to form a near-field electromagnetic wave which propagates along the surface of the metal; under the condition of satisfying wave vector matching (conservation of momentum), the energy of the incident electromagnetic field is effectively converted into collective vibration energy of free electrons on the metal surface, and a so-called surface plasmon is formed. SPPs have properties of sub-wavelength, near-field local and surface enhancement, environmental sensitivity, etc., and are often used in microscopic imaging based on these excellent properties. However, there is a great limitation in the use of surface plasmon microscopy imaging systems for imaging detection of small particles below 100nm in diameter: because SPPs propagating along the metal surface interfere with scattered light, the generated interference fringes greatly influence the spatial resolution of imaging; meanwhile, for particles with diameters below 50nm, the scattered signal intensity is very weak, and the particles are easily submerged in background noise. These problems restrict the imaging and detection of small-particle-size particles by surface plasmon imaging systems, and therefore a solution is needed.
Disclosure of Invention
The invention aims to overcome the problem of low signal-to-noise ratio of the traditional surface plasmon microscopy imaging system, and provides a surface plasmon imaging system based on differential denoising, which can perform high signal-to-noise ratio imaging on nanoparticles with the diameter as low as 22nm and has the particle size resolution of 10 nm. Meanwhile, the required substrate is easy to prepare, the imaging process is not damaged, and the method is suitable for various biochemical samples, so that the microscopic imaging method has wide application value.
The technical scheme for realizing the purpose is as follows:
a differential denoising-based surface plasmon imaging system, the imaging system comprising: the device comprises a rotary illumination system, a tube lens, a beam splitter, an oil immersion microscope objective, a gold film substrate, a high-precision displacement platform and an imaging light path; one laser beam is collimated by the collimating lens, is modulated by the rotary lighting system into linearly polarized light in any direction, and is emitted; the emergent light beam passes through the tube lens and the beam splitter and is focused to the back focal plane of the oil immersion microscope objective; the focused light beam passes through an oil immersion microscope and then irradiates the gold film substrate in a parallel light mode; through the modulation of a rotary illumination system, parallel light beams can be incident to the gold film substrate at the excitation angle of the surface plasmon and the TM polarization direction, so that the surface plasmon is excited on a gold film-air interface; when the surface plasmon propagating along the interface irradiates a sample on the gold film substrate, the surface plasmon and the sample interfere with scattered light of the sample to generate interference fringes; the emergent angle of the light beam is modulated by a rotary illumination system, so that a focused light spot on a back focal plane of the light beam rotates along a circular track with a fixed radius, and rotary illumination of the light beam on a sample plane is realized; meanwhile, the high-precision displacement platform for supporting the gold film substrate moves in a fixed step length along the horizontal direction, and the sample moves and stays along with the high-precision displacement platform after moving one step length; collecting scattered light leaked from the gold film substrate through an oil immersion microscope objective, transmitting the scattered light through a beam splitter, entering an imaging light path in a parallel light mode, and carrying out exposure imaging on a gap imaging light path where the sample stays each time to obtain images of a series of samples; subtracting the gray values of two adjacent images in the series of images to eliminate noise caused by defects in the system, such as noise introduced by uneven light spots, pollutants on the surface of an optical element and the like, and obtaining a series of corresponding difference images; extracting a region with the size of 400 × 400 pixels by taking the image of the sample as the center in the differential image to obtain an extracted image; and superposing and averaging the gray values of all the extracted images, and eliminating noise which changes along with time in the system, such as noise introduced by the fluctuation of the light spot intensity along with time, so as to obtain the finally processed image. Through the processing of the later-stage differential denoising algorithm, the noise of the image is eliminated, and the signal-to-noise ratio of the surface plasmon imaging system is greatly improved.
The rotary illumination system is composed of a reflecting vibrating mirror, a polarizer and a half-wave plate. Through the modulation of the two reflecting vibration mirrors, the emergent angle of the light beam can be changed, and finally the light beam is incident to the gold film substrate by taking the surface plasmon excitation angle as a radial angle and rotates along the azimuth angle direction. The polarizer is used for changing the light beam into linearly polarized light, the polarization direction of the polarized light can be changed by rotating the half-wave plate until the polarization direction is the polarization direction corresponding to the TM mode relative to the gold film substrate, and therefore excitation of surface plasmons is achieved.
The oil immersion microscope objective has two functions, and is firstly used for converting focused light into parallel light to irradiate the gold film substrate and exciting surface plasmons on the substrate; and then used to collect scattered light that leaks off the gold film.
The gold film substrate consists of a bottom layer cover glass, a middle layer of 3nm titanium and an upper layer of 45nm gold. The bottom cover glass is used for providing wave vector matching when surface plasmons are excited, the middle layer of 3nm titanium is used as an adhesion layer, so that the upper layer of gold film does not fall off, and the upper layer of gold film is used for exciting the surface plasmons.
The high-precision displacement platform can perform nano-scale high-precision displacement, and sends a trigger signal to the detector after the displacement platform is in place, so that the detector starts exposure.
The imaging light path comprises an analyzer, an imaging tube lens and a detector. The polarization direction of the analyzer is orthogonal to the polarization direction of the polarizer, so that most of reflected light can be blocked, and background noise is suppressed. The imaging tube lens focuses the scattered light to the detector chip to realize imaging. The detector is controlled by a trigger signal from the high-precision displacement platform to carry out exposure and photographing.
The principle of the technical scheme of the invention is as follows:
in the microscopic imaging process, the background noise distribution of imaging is not changed by slightly moving the substrate, so that the background noise can be removed by subtracting two pictures taken before and after the moving substrate. Because of the difference, the step length of each movement must be larger than the maximum length of the sample along the moving direction, so as to ensure that the images of the sample in the two pictures are not eliminated due to the overlapping part after the difference is made. However, the conventional surface plasmon microscopy imaging system enlarges the image of the sample having a particle size of less than 100nm to several tens of micrometers due to the presence of interference fringes. In order to reduce the size of the image of the sample, a rotary illumination system is adopted, an illumination light beam rotates for more than one circle at a high speed along the azimuth direction within one exposure time, interference fringes obtained by incidence at different azimuths are different in orientation, the interference fringes in each orientation are superposed and averaged on a detector, the interference fringes are eliminated, the size of the image of the sample is reduced to hundreds of nanometers, and therefore the high-precision displacement platform can be translated in a small step size. In order to eliminate the noise caused by the change of the illumination light intensity distribution along with the time, a row of continuous images are shot for difference, and the images after difference are averaged to eliminate the noise, so that the signal to noise ratio of the imaging is greatly improved.
Compared with the prior imaging technology, the invention has the advantages that:
the invention enables the imaging system to have extremely high signal-to-noise ratio: polystyrene spheres with particle sizes as low as 22nm can be imaged clearly, while the limit of a conventional surface plasmon microscope is to image polystyrene spheres with particle sizes of 50-100 nm.
Drawings
FIG. 1 is a schematic structural diagram of a differential denoising-based surface plasmon imaging system according to the present invention;
FIG. 2 is a flow chart of a differential denoising method;
FIG. 3 is a transmission electron micrograph and a surface plasmon imaging chart of polystyrene spheres with diameters of 22nm, 31nm, 41nm, 51nm, 61nm and 81nm, respectively, wherein FIG. 3a is a transmission electron micrograph of polystyrene spheres with different diameters, and the scalebar length is 50 nm; FIG. 3b is a graph of surface plasmon images of polystyrene beads of different diameters obtained using the system, with scalebar length of 1 micron; fig. 3c is an intensity cross-section drawn along the dashed line in fig. 3 b.
The reference numerals have the meanings: 1 is a rotary lighting system; 2 is a tube lens; 3 is a beam splitter; 4 is an oil immersion microscope objective; 5 is a gold film substrate; 6, a high-precision displacement platform; 7 is an imaging light path; 8 is a first galvanometer; 9 is a second galvanometer; 10 is a polarizer; 11 is a half-wave plate; 12 is an analyzer; 13 is an imaging tube lens; and 14 is a detector.
Detailed Description
The invention is described in further detail below with reference to the figures and the detailed description.
Referring to fig. 1-2, a surface plasmon imaging system based on differential denoising includes: the device comprises a rotary illumination system 1, a tube lens 2, a beam splitter 3, an oil immersion microscope objective 4, a gold film substrate 5, a high-precision displacement platform 6, an imaging light path 7, a first vibrating mirror 8, a second vibrating mirror 9, a polarizer 10, a half-wave plate 11, an analyzer 12, an imaging tube lens 13 and a detector 14.
The rotary illumination system 1 is composed of a first vibrating mirror 8, a second vibrating mirror 9, a polarizer 10 and a half-wave plate 11. Through the modulation of the two reflecting vibration mirrors, the emergent angle of the light beam can be changed, and finally the light beam is incident to the gold film substrate 5 by taking the surface plasmon excitation angle as a radial angle and rotates along the azimuth angle direction. The polarizer 10 is used for changing the light beam into linearly polarized light, and the polarization direction of the polarized light can be changed by rotating the half-wave plate 11 until the polarization direction is the polarization direction corresponding to the TM mode relative to the gold film substrate 5, so that the excitation of the surface plasmon is realized.
The imaging optical path 7 comprises an analyzer 12, an imaging tube lens 13 and a detector 14. The polarization direction of the analyzer 12 is orthogonal to the polarization direction of the polarizer 10, so that most of reflected light can be blocked, and background noise is suppressed. The imaging tube lens 13 focuses the scattered light to the detector 14 chip, so as to realize imaging. The detector 14 is controlled by a trigger signal from the high-precision displacement platform 6 to carry out exposure photographing.
The oil immersion microscope objective 4 has two functions, and is used for converting focused light into parallel light to irradiate the gold film substrate 5 and exciting surface plasmons on the substrate; and then used to collect scattered light that leaks off the gold film.
The gold film substrate 5 is composed of a bottom layer cover glass, a middle layer of 3nm titanium and an upper layer of 45nm gold. The bottom cover glass is used for providing wave vector matching when surface plasmons are excited, the middle layer of 3nm titanium is used as an adhesion layer, so that the upper layer of gold film does not fall off, and the upper layer of gold film is used for exciting the surface plasmons.
The high-precision displacement platform 6 can perform nano-scale high-precision displacement, and sends a trigger signal to the detector 14 after the displacement platform is in place, so that the detector starts to be exposed.
One beam of laser after being collimated by the collimating lens is modulated into linearly polarized light in any direction sequentially through a first vibrating mirror 8, a second vibrating mirror 9, a polarizer 10 and a half-wave plate 11 in the rotary lighting system 1 and is emitted at a specific angle; the emergent light beam passes through the tube lens 2 and the beam splitter 3 and is focused to the back focal plane of the oil immersion microscope objective 4; the focused light beam passes through an oil immersion microscope objective 4 and then irradiates a gold film substrate 5 in a parallel light mode; by controlling the angles of the first galvanometer 8 and the second galvanometer 9, parallel light beams are made to be incident to the gold film substrate 5 by taking a fixed azimuth angle and a surface plasmon excitation angle as radial angles, so that the one-way illumination function of a traditional surface plasmon imaging system on a sample is realized, and the function is shown in fig. 2A; rotating the parallel light beams along the azimuth direction and making the radial angle be the surface plasmon excitation angle to be incident on the gold film substrate 5 to realize the rotating illumination of the sample, as shown in fig. 2B; by controlling the high-precision displacement platform 6, the gold film substrate 5 and the sample placed on the gold film substrate move in a fixed step length along the horizontal direction, and pause is performed when the gold film substrate moves by one step length, at the moment, the detector 14 performs exposure photographing, and a series of sample images are obtained, as shown in fig. 2C; subtracting the front and back of two adjacent pictures in the series of pictures to obtain corresponding difference pictures, which are shown in figure 2D; extracting 400 × 400 pixel regions around the sample as the center in the differential image by using a computer program to obtain an extraction image, which is shown in fig. 2E; and finally, superposing the gray values of all the extracted images and dividing the superposed gray values by the number of the extracted images to obtain a final image, which is shown in figure 2F. As can be seen by comparing fig. 2A and 2F, the system has a higher signal-to-noise ratio than the conventional surface plasmon imaging system.
Referring to FIG. 3, graph A is a transmission electron microscope image of polystyrene beads with diameters of 22nm, 31nm, 41nm, 51nm, 61nm and 81nm, respectively; the B picture is an image obtained by imaging a corresponding small ball through a differential denoising-based surface plasmon imaging system, and shows that the system can image particles with low refractive index with the diameter of 22nm and has the diameter resolution of 10 nm; the C image is an intensity section of the dotted line position in the B image, and shows that the imaging by the system can obtain extremely high signal-to-noise ratio.
Parts of the invention not described in detail are well known in the art. The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and the preferred embodiments are not exhaustive and do not limit the invention to the precise embodiments described. Various modifications and improvements of the technical solution of the present invention may be made by those skilled in the art without departing from the spirit of the present invention, and the technical solution of the present invention is to be covered by the protection scope defined by the claims.
Claims (6)
1. The utility model provides a surface plasmon imaging system based on difference is denoised which characterized in that: the imaging system includes: the device comprises a rotary illumination system (1), a tube lens (2), a beam splitter (3), an oil immersion microscope objective (4), a gold film substrate (5), a high-precision displacement platform (6) and an imaging light path (7); wherein the content of the first and second substances,
one laser beam after being collimated by the collimating lens is modulated into linearly polarized light in any direction by the rotary lighting system (1) and is emitted; the emergent light beam passes through the tube lens (2) and the beam splitter (3) and is focused to the back focal plane of the oil immersion microscope objective (4); the focused light beam passes through an oil immersion microscope objective (4) and then irradiates a gold film substrate (5) in a parallel light mode; the parallel light beams can be incident to the gold film substrate (5) at the excitation angle of the surface plasmon and the TM polarization direction through the modulation of the rotary illumination system (1), so that the surface plasmon is excited on a gold film-air interface; when the surface plasmon propagating along the interface irradiates a sample on the gold film substrate (5), interference fringes are generated by interference of scattered light of the sample; the emergent angle of the light beam is modulated by the rotary illumination system (1), so that a focused light spot of the light beam on a back focal plane rotates along a circular track with a fixed radius, and rotary illumination of the light beam on a sample plane is realized; meanwhile, the high-precision displacement platform (6) for supporting the gold film substrate (5) moves in a fixed step length along the horizontal direction, and the sample moves and stays after moving one step length; scattered light leaked from the gold film substrate (5) is collected through the oil immersion microscope objective (4), and after being transmitted through the beam splitter (3), the scattered light enters the imaging light path (7) in a parallel light mode, and is exposed and imaged in the gap imaging light path where the sample stays each time to obtain a series of images of the sample; subtracting the gray values of two adjacent images in the series of images to eliminate noise caused by defects in the system and obtain a series of corresponding difference images; extracting a region with the size of 400 × 400 pixels by taking the image of the sample as the center in the differential image to obtain an extracted image; and superposing the gray values of all the extracted images to average, and eliminating noise which changes along with time in the system to obtain the finally processed image.
2. The differential denoising-based surface plasmon imaging system according to claim 1, wherein the rotating illumination system (1) is composed of a first galvanometer (8), a second galvanometer (9), a polarizer (10) and a half-wave plate (11); the emergent angle of the light beam can be changed through the modulation of the two reflecting vibration mirrors, and finally the light beam is incident to the gold film substrate (5) by taking the surface plasmon excitation angle as a radial angle and rotates along the azimuth angle direction; the polarizer (10) is used for changing the light beam into linear polarization, the polarization direction of the polarized light can be changed by rotating the half-wave plate (11) until the polarization direction is the polarization direction corresponding to the TM mode relative to the gold film substrate (5), and therefore excitation of surface plasmons is achieved.
3. The differential denoising-based surface plasmon imaging system according to claim 1, wherein the oil immersion microscope objective (4) has two functions, firstly converting the focused light into parallel light to irradiate the gold film substrate (5) and exciting surface plasmons on the substrate; and then used to collect scattered light that leaks off the gold film.
4. The differential denoising-based surface plasmon imaging system according to claim 1, wherein the gold film substrate (5) is composed of a bottom cover glass, a middle layer of 3nm titanium and an upper layer of 45nm gold; the bottom cover glass is used for providing wave vector matching when surface plasmons are excited, the middle layer of 3nm titanium is used as an adhesion layer, so that the upper layer of gold film does not fall off, and the upper layer of gold film is used for exciting the surface plasmons.
5. The differential denoising-based surface plasmon imaging system according to claim 1, wherein the high precision displacement platform (6) can perform high precision displacement of nanometer scale, and sends a trigger signal to the detector (14) after being in place, so that the detector starts exposure.
6. The differential denoising-based surface plasmon imaging system according to claim 1, wherein the imaging optical path (7) comprises an analyzer (12), an imaging tube mirror (13) and a detector (14); the transmission vibration direction of the analyzer (12) is orthogonal to the transmission vibration direction of the polarizer (10); the imaging tube lens (13) focuses the scattered light to a detector (14) chip to realize imaging; the detector (14) is controlled by a trigger signal from the high-precision displacement platform (6) to carry out exposure and photographing.
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