Single nanoparticle bright and dark field confocal imaging and spectral measurement system

Abstract

The invention belongs to the technical field of microscopic imaging and spectral measurement, and particularly relates to a bright and dark field confocal imaging and spectral measurement system for single nanoparticles. In order to realize single-particle bright and dark field imaging switching and single-particle dark field scattering imaging and spectrum synchronous real-time measurement, the system realizes bright and dark field imaging mode switching through a bright field light splitting plain film and a dark field hollow reflector plain film. The piezoelectric three-dimensional displacement table is controlled to carry out two-dimensional plane scanning and axial position adjustment on a sample, and a cross-shaped slit is combined to limit a transmission area of two-dimensional plane reflected light or scattered light, so that single-particle positioning is realized, and interference of adjacent particles is shielded. The positioned single-particle reflected light or scattered light is split by the beam splitter prism, a reflected light beam enters the color CCD to realize bright and dark field imaging, a transmitted light beam is divided into 0-level diffraction and 1-level diffraction through the transmission-type grating and is focused to the EMCCD, the 0-level light is single-particle imaging, and the 1-level diffraction is used for dark field scattering spectrum measurement.

Description

Technical Field

[0001] The present invention belongs to the technical field of microscopic imaging and spectral measurement, and particularly relates to a bright and dark field confocal imaging and spectral measurement system for single nanoparticles. Background Art

[0002] With the rapid development of nanotechnology, the precise positioning, morphology characterization and synchronous analysis of optical properties of single metal nanoparticles (such as gold and silver nanoparticles) have become the core requirements in the fields of nanodevice design, biosensing, etc. Although traditional bright field microscopy can achieve fast imaging, the low optical contrast between metal nanoparticles and the medium environment makes it difficult to identify single particles; dark field microscopy can greatly suppress background light and obtain the imaging of the scattering signal of single metal nanoparticles, but its wide field of view imaging mode is difficult to obtain the scattering spectrum of single metal nanoparticles. Therefore, developing an integrated system that can in-situ switch between bright and dark field imaging modes, synchronously obtain the spatial coordinates, imaging and spectral information of single particles, has low cost, and has low light damage characteristics and high spatial resolution can make up for the deficiencies of current technologies. Summary of the Invention

[0003] In view of the above problems, the present invention provides a bright and dark field confocal imaging and spectral measurement system for single nanoparticles. This system can switch between bright field imaging and dark field imaging modes in the same system according to experimental requirements. In the dark field imaging mode, it can accurately locate single nanoparticles, measure the dark field scattering spectrum, and shield the interference of the scattering of adjacent particles on the measurement of the dark field scattering spectrum of the nanoparticle to be measured. It can obtain the dark field imaging and dark field scattering spectrum of single particles, and realize the synchronous real-time detection of the dark field scattering imaging and scattering spectrum evolution of single particles over time.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A bright and dark field confocal imaging and spectral measurement system for single nanoparticles includes a white light collimation module, a bright and dark field switching module, an excitation scanning module, a single particle positioning module, and an imaging and spectral measurement module.

[0006] The white light collimation module sequentially includes a halogen lamp, a filter, a first reflector, a second reflector, and a collimating lens in the light propagation direction. The filter serves to change the color temperature and isolate the influence of stray light in the environment on the imaging quality. The two reflectors adjust the position and imaging uniformity of the white light before and after the objective lens. The collimating lens collimates the incident light, and the collimated light enters the bright and dark field switching module.

[0007] The bright and dark field switching module includes a bright field beam splitter flat plate, a dark field hollow reflection flat plate, and a light shielding sheet. The bright field beam splitter flat plate is a 5:5 beam splitter. The dark field hollow reflection flat plate is an elliptical hollow mirror. The light shielding sheet is a black circular light shielding sheet.

[0008] Furthermore, in the bright-field imaging mode, an incident white light is reflected by a bright-field beam splitter flat plate into an excitation scanning module to irradiate a sample. The reflected light of the sample passes through the bright-field beam splitter flat plate and is transmitted to an imaging and spectral measurement module.

[0009] Furthermore, in the dark-field imaging mode, the bright-field beam splitter flat plate is replaced by a dark-field hollow reflection flat plate through a magnetic block. A light-shielding sheet is placed in front of the dark-field hollow reflection flat plate to block the central area of the light spot, forming an annular light spot. The annular light spot is reflected by the edge of the dark-field hollow reflection flat plate into the excitation scanning module to irradiate the sample. The scattered light of the sample passes through the center of the dark-field hollow reflection flat plate and enters the imaging and spectral measurement module.

[0010] The excitation scanning module includes an objective lens, a sample, and a piezoelectric three-dimensional displacement stage. The white light reflected by the bright and dark field switching module is focused on the sample through the objective lens. The sample is placed on the piezoelectric three-dimensional displacement stage, and the piezoelectric three-dimensional displacement stage is controlled to move to perform two-dimensional plane scanning and axial position adjustment on the sample.

[0011] The single-particle positioning module includes a third mirror, a first lens, a cross slit, and a second lens. The reflected light or scattered light of the sample collected by the objective lens passes through the bright and dark field switching module, is reflected by the third mirror, and is focused by the first lens to the center of the cross slit. The cross slit is used to limit the transmission area of the reflected light or scattered light in the two-dimensional plane, position the single-particle sample, and shield the interference of adjacent particles. The second lens collimates the reflected light or scattered light of the sample and enters the imaging and spectral measurement module.

[0012] The imaging and spectral measurement module includes a beam splitting prism, a third lens, a color CCD, a transmission grating, a fourth lens, and an EMCCD. The reflected light or scattered light of the sample (particle) positioned by the single-particle positioning module is split into a transmitted beam and a reflected beam by the beam splitting prism. In the bright-field imaging mode, the reflected beam is focused on the color CCD through the third lens to achieve bright-field imaging. In the dark-field imaging mode, the transmitted beam is sequentially split into a 0th order and a 1st order diffraction by the transmission grating, and is focused on the EMCCD through the fourth lens. The 0th order light is for single-particle imaging, and the 1st order diffraction is for dark-field scattering spectrum analysis.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] First, by controlling the piezoelectric three-dimensional displacement stage to perform two-dimensional plane scanning and axial position adjustment on the sample, the single particle to be measured can be adjusted to the focusing position of the objective lens; combined with spatial filtering using a two-dimensionally adjustable cross slit, the single particle to be measured on the sample can be accurately positioned, and the interference of adjacent particle scattering on the dark-field scattering spectrum measurement of the single particle to be measured can be shielded.

[0015] Second, on the basis of the above single-particle positioning, the dark-field scattered light of a single particle is decomposed into 0th-order and 1st-order diffractions by a transmission grating. With the aid of an EMCCD detector, the dark-field imaging of a single particle and the measurement of the dark-field scattering spectrum can be obtained simultaneously in one frame of image, realizing the real-time detection of the dark-field scattering imaging and scattering spectrum of a single particle over time. Description of the Drawings

[0016] Figure 1 Schematic structural diagram of a bright and dark field confocal imaging and spectrum measurement system for a single nanoparticle provided by the present invention;

[0017] Figure 2 Bright field imaging of gold nanoparticles;

[0018] Figure 3 Dark field imaging of gold nanoparticles;

[0019] Figure 4 0th-order imaging and 1st-order diffraction pattern of a single gold nanoparticle;

[0020] Figure 5 Imaging of a single gold nanoparticle located by a cross slit;

[0021] Figure 6 Dark field scattering spectrum of a single gold nanoparticle obtained by using 1st-order diffraction. Detailed Embodiments

[0022] In order to further elaborate the technical solution of the present invention, the present invention will be described below by way of examples.

[0023] As shown in the attached Figure 1 figure, this embodiment provides a bright and dark field confocal imaging and spectrum measurement system for a single nanoparticle, which is used to realize the functions of bright and dark field imaging, positioning and dark field scattering spectrum detection of a single nanoparticle.

[0024] This system includes a white light collimation module, a bright and dark field switching module, an excitation scanning module, a single particle positioning module, and an imaging and spectrum measurement module.

[0025] The white light collimation module provides a collimated illumination light source for bright and dark field imaging. The white light emitted by the halogen lamp 1 sequentially passes through the filter 2, the first mirror 3, the second mirror 4, and the collimating lens 5 to the bright and dark field switching module 6. The filter 2 described in this embodiment is used to change the color temperature and isolate the influence of stray light in the environment on the imaging quality.

[0026] The bright and dark field switching module 6 is used to switch between bright and dark field imaging modes. It includes a bright field beam splitter flat mirror 18, a dark field hollow reflection flat mirror 19, and a light shield 20. The bright field beam splitter flat mirror 18 is a 5:5 beam splitter. The dark field module includes a light shield 20 and a dark field hollow reflection flat mirror 19. The dark field hollow reflection flat mirror 19 is an elliptical hollow reflector. The light shield 20 is a black circular light blocking sheet.

[0027] Attached Figure 2 is the bright field imaging of gold nanoparticles. The specific implementation method is as follows: The bright field beam splitter flat mirror 18 reflects white light into the objective lens 7 and focuses it on the sample 8. The reflected light of the sample 8 is collected by the objective lens 7, passes through the bright field beam splitter flat mirror 18, and is transmitted to the third reflector 9. Then, it passes through the first lens 10, the cross slit 21, the second lens 11 to the beam splitter prism 12, and is divided into a transmitted beam and a reflected beam. The reflected beam passes through the third lens 16 to the color CCD 17 to achieve bright field imaging.

[0028] Attached Figure 3 is the dark field imaging of gold nanoparticles. The specific implementation method is as follows: Replace the bright field beam splitter flat mirror 18 in the bright and dark field switching module 6 with the dark field hollow reflection flat mirror 19 through a magnetic block, and insert a light shield 20 on the optical path between the collimating lens 5 and the hollow reflection flat mirror 19. The white light passes through the light shield 20 to block the central area of the light spot, forming an annular light spot. The annular light spot is reflected by the edge of the dark field hollow reflection flat mirror 19 and enters the objective lens 7 to be focused to form an annular illumination light spot on the sample 8. The scattered light of the sample 8 is collected by the objective lens 7, passes through the center of the hollow reflection flat mirror 19, is reflected by the third reflector 9, and then passes through the first lens 10, the cross slit 21, the second lens 11 to the beam splitter prism 12, which is divided into a transmitted beam and a reflected beam. The reflected beam passes through the third lens 16 to the color CCD 17 to achieve dark field imaging; the transmitted beam passes through the transmission grating 13 and the fourth lens 14 to the EMCCD 15 in sequence to achieve 0th and 1st order diffraction imaging (attached Figure 4 ), and the measurement of the dark field scattering spectrum (attached Figure 6 ).

[0029] The functions of the first reflector 3 and the second reflector 4 in this embodiment are to control the uniformity of the incident annular light spot before entering the objective lens 7 by adjusting the pitch of the first reflector 3, and to control the uniformity of the dark field annular light spot focused on the sample 8 through the objective lens 7 by adjusting the pitch of the second reflector 4, ensuring that clear dark field imaging can be presented on the color CCD 17.

[0030] The single-particle positioning module described in this embodiment includes a third reflector 9, a first lens 10, a cross slit 21, and a second lens 11. The objective lens 7 collects the reflected light or scattered light of the sample 8, which passes through the bright and dark field switching module 6, is reflected by the third reflector 9, and is focused by the first lens 10 onto the center of the cross slit 21. The cross slit 21 is used to limit the transmission area of the reflected light or scattered light in the two-dimensional plane, position the single-particle sample, and shield the interference of adjacent particles. The second lens 11 collimates the light spot and enters the imaging and spectral measurement module.

[0031] The excitation scanning module described in this embodiment assists in realizing single-particle positioning, imaging, and spectral measurement. The white light reflected by the bright and dark field switching module 6 is focused onto the sample 8 through the objective lens 7. The sample 8 is placed on the piezoelectric three-dimensional displacement stage 22, and the piezoelectric three-dimensional displacement stage 22 is manipulated to move for two-dimensional plane scanning and axial position adjustment of the sample. The adjustable range of the piezoelectric three-dimensional displacement stage 22 is 100*100*100 μm; combined with the two-dimensionally adjustable cross slit 21 for spatial filtering, single-particle imaging is observed on the color CCD 17 (attached Figure 5 ), and the single particle to be measured on the sample can be accurately positioned.

[0032] The above shows and describes the main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to include all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

[0033] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A bright and dark field confocal imaging and spectral measurement system for single nanoparticles, characterized in that: It includes a white light collimation module, a bright and dark field switching module (6), an excitation scanning module, a single particle positioning module, and an imaging and spectral measurement module.

2. A single nanoparticle bright and dark field confocal imaging and spectral measurement system according to claim 1, characterized in that: The white light collimating module comprises a halogen lamp (1), a filter (2), a first reflector (3), a second reflector (4) and a collimating lens (5) in sequence according to the light propagation direction; the collimated light after passing through the collimating lens (5) enters a bright and dark field switching module (6).

3. The bright-field and dark-field confocal imaging and spectral measurement system of a single nanoparticle according to claim 1, characterized in that: The bright and dark field switching module (6) comprises a bright field beam splitter plate (18), a dark field hollow reflector plate (19) and a light shielding plate (20); the bright field beam splitter plate (18) is a 5:5 beam splitter plate, the dark field hollow reflector plate (19) is an elliptical hollow reflector, and the light shielding plate (20) is a black circular light shielding plate.

4. A single nanoparticle bright and dark field confocal imaging and spectral measurement system according to claim 3, characterized in that: The bright and dark field switching module (6) is used to switch between bright and dark field imaging modes. In the bright field imaging mode, a bright field spectroscopic plate (18) is used to reflect incident white light into an excitation scanning module to illuminate a sample (8). The reflected light of the sample (8) passes through the bright field spectroscopic plate (18) and is transmitted to the imaging and spectral measurement module. In the dark field imaging mode, the bright field spectroscopic plate (18) is replaced with a dark field hollow reflection plate (19) through a magnetic suction block. A light shielding plate (20) is placed in front of the dark field hollow reflection plate (19) to shield the center area of ​​the light spot, forming an annular light spot. The annular light spot is reflected from the edge of the dark field hollow reflection plate (19) into the excitation scanning module to illuminate the sample (8). The scattered light of the sample (8) passes through the center of the dark field hollow reflection mirror (19) and enters the imaging and spectral measurement module.

5. The bright and dark field confocal imaging and spectral measurement system of a single nanoparticle according to claim 1, characterized in that: The excitation scanning module comprises an objective lens (7), a sample (8), and a piezoelectric three-dimensional displacement stage (22); the white light reflected by the bright and dark field switching module (6) is focused onto the sample (8) through the objective lens (7); the sample (8) is placed on the piezoelectric three-dimensional displacement stage (22); the piezoelectric three-dimensional displacement stage (22) is controlled to move to perform two-dimensional plane scanning and axial position adjustment on the sample (8).

6. The bright-field and dark-field confocal imaging and spectral measurement system of a single nanoparticle according to claim 1, characterized in that: The single particle positioning module comprises a third reflector (9), a first lens (10), a cross slit (21), and a second lens (11); the reflected light or scattered light of the sample (8) collected by the objective lens (7) passes through the bright and dark field switching module (6), is reflected by the third reflector (9), and is focused by the first lens (10) to the center of the cross slit (21); the cross slit (21) is used to limit the transmission area of ​​the reflected light or scattered light in the two-dimensional plane, locate the single particle sample, and shield the interference of adjacent particles; the second lens (11) collimates the reflected light or scattered light of the sample (8) and enters the imaging and spectral measurement module.

7. The bright and dark field confocal imaging and spectral measurement system of a single nanoparticle according to claim 1, characterized in that: The imaging and spectral measurement module comprises a beam splitter prism (12), a third lens (16), a color CCD (17), a transmission grating (13), a fourth lens (14) and an EMCCD (15). The reflected light or scattered light of the sample (8) positioned by the single particle positioning module is divided into a transmitted light beam and a reflected light beam by the beam splitter prism (12). In a bright field imaging mode, the reflected light beam is focused onto the color CCD (17) through the third lens (16) to achieve bright field imaging. In a dark field imaging mode, the transmitted light beam is sequentially divided into 0th order and 1st order diffraction through the transmission grating (13), and is focused onto the EMCCD (15) through the fourth lens (14). The 0th order light is for single particle imaging, and the 1st order diffraction is used for dark field scattering spectrum measurement.

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