A plasmonic coupled interfacial in-situ spectroscopy and imaging test system

Abstract

The application provides a plasmon coupling surface interface in-situ spectrum and imaging test system, which records reflected light intensity or spectrum while changing laser incidence angle, and when the incidence angle of the laser is just in the range of the optical near field and excites surface plasmon polariton of a silver film in a prism coupling manner, the minimum point of the reflected light intensity is observed, that is, surface plasmon resonance, the incidence light is kept to excite the sample at the resonance angle, and at this time, the target molecule system on the surface of the silver film scatters and absorbs the electromagnetic surface wave bound in the near field range, and re-emits the light signal to the far field. The system realizes in-situ combination of surface plasmon resonance, surface enhanced Raman scattering spectrum, total internal reflection fluorescence spectrum and imaging technology by collecting the light signals.

Description

Technical Field

[0001] This invention belongs to the field of surface and interface spectral analysis, specifically relating to a plasmonic-coupled in-situ spectral and imaging testing system for surfaces and interfaces. Background Technology

[0002] The study and understanding of the physicochemical processes at interfaces are of great significance for advancing many disciplines, including photocatalysis, sensing and detection, and integrated optoelectronic devices. Various techniques have been established in the exploration of interface science, such as electron microscopy, scanning probe microscopy, photoelectron spectroscopy, and electrochemical techniques, which provide information about interfaces at different levels. Spectroscopy, as an important research tool, has been widely and deeply developed in interface science due to its advantages of low damage, in-situ operation, flexible operating environment, and access to information about the structure of reactants.

[0003] Raman spectroscopy and fluorescence spectroscopy are the two most widely used spectroscopic techniques in interface analysis. Raman spectroscopy, as a type of fingerprint spectroscopy, is related to the vibrational modes of molecules in a chemical environment. By analyzing the peak position, peak intensity, and full width at half maximum (FWHM) of Raman spectra, the structural and orientational changes of molecules at the interface can be obtained. Furthermore, with the development of surface-enhanced Raman spectroscopy (SMR), the sensitivity of Raman spectroscopy has been significantly improved. By constructing suitable Raman-enhancing substrates and selecting efficient excitation and acquisition methods, single-molecule level Raman analysis and detection have become a reality. Fluorescence spectroscopy and imaging are highly versatile techniques in the study of physicochemical processes at interfaces. On the one hand, fluorescence spectroscopy reflects the excited-state information of substances, and by analyzing fluorescence spectra, indirect evidence of intermolecular interactions can be obtained. On the other hand, fluorescence labeling methods can be used for highly sensitive detection and tracking of physicochemical processes at interfaces, such as obtaining the spatial distribution of target molecules and detecting binding events between molecules and substrates.

[0004] The inherent characteristics of interface problems place high demands on analytical methods. Firstly, interface research focuses on the micro- and nanoscale regions near the interface. To shield against interference from signals or background noise from outside the interface, the chosen techniques should possess high spatial resolution in the direction perpendicular to the interface. Secondly, since the objects analyzed in interface problems are often thin films composed of single or few layers of molecules, the analytical techniques must have sufficiently high sensitivity to acquire signals from low concentrations of the target substance. Finally, the physicochemical processes occurring at interfaces are highly complex. To resolve these problems, a combination of analytical techniques is needed to characterize the target in different dimensions.

[0005] Surface plasmon polaritons offer an effective solution to overcome the aforementioned problems. First, the electromagnetic field has a very small penetration depth in the direction perpendicular to the metal-dielectric interface, thus effectively limiting the spectral detection range to the interface region. Second, due to the optical field modulation capability of plasmon polaritons, the excitation field energy can be highly concentrated in the hot spot region. This electromagnetic field effect can significantly enhance the intensity of Raman and fluorescence spectral signals.

[0006] Existing technologies, including patent CN1657914A and academic articles (Liu, Yu, et al. Review of Scientific Instruments 81.3(2010):036105.), establish an in-situ SPR-SERS testing device. This invention differs from these in three aspects: 1. It uses the prism reflection side instead of the air transmission side to collect Raman signals, thus effectively utilizing the directional emission effect of plasmons. 2. By adding a polarizing beam-splitting prism to the reflecting arm, it can simultaneously measure both S and P polarized light signals, which can be applied to studies related to interface molecular orientation. 3. In addition to testing SPR and SERS, this invention can also perform total internal reflection fluorescence spectroscopy and imaging tests, realizing the combined use of SPR, SERS, and TIRF technologies. 4. In this invention, the excitation light is coupled into the system via optical fiber, rather than the laser being directly fixed to the incident arm, thus allowing for convenient switching of the excitation wavelength. Summary of the Invention

[0007] To address the problems existing in surface and interface spectral analysis techniques, and combining the advantages of surface plasmon resonance, surface-enhanced Raman spectroscopy, and total internal reflection fluorescence microscopy, this invention constructs an in-situ testing platform for plasmon-coupled surface and interface fluorescence and Raman spectroscopy with incident angle control. This platform records the intensity or spectrum of reflected light while changing the laser incident angle. When the incident laser angle is precisely adjusted to excite surface plasmon polaritons on a silver (or gold) film in the near-field optical range via prism coupling, the reflected light intensity reaches its lowest point, a state known as surface plasmon resonance. By maintaining the incident light at the resonance angle to excite the sample, the target molecular system modified on the silver film surface scatters and absorbs the electromagnetic surface waves confined in the near-field range, re-emitting the light signal to the far field. This invention's device achieves in-situ coupling of surface plasmon resonance, surface-enhanced Raman scattering spectroscopy, total internal reflection fluorescence spectroscopy, and imaging techniques by collecting these light signals.

[0008] The present invention discloses a plasmonic coupling in-situ spectral and imaging testing system for surfaces and interfaces, comprising a laser coaxial adjustment and fiber optic coupling system, a frame and a rotating arm drive device, a variable angle incident arm and a photoexcitation system, a variable angle reflection arm and a light signal acquisition system, a sample stage and a prism mounting frame, and a microscopic spectral acquisition and imaging system; a cylindrical prism is mounted on the sample stage and the prism mounting frame.

[0009] The laser coaxial adjustment and fiber coupling system includes: a long-pass dichroic mirror, a set of lasers of different wavelengths, a fiber coupler, an optical fiber, and a reflector. The long-pass dichroic mirror combines a set of lasers of different wavelengths into a beam, which is reflected by the reflector and coupled into the optical fiber by the fiber coupler, and finally transmitted to the variable angle incident arm and optical excitation system.

[0010] The variable angle incident arm and optical excitation system include: fiber optic flange, aspherical lens, linear polarizer, half-wave plate, adjustable aperture, and focusing objective; the light beam coupled from the fiber to free space is first collimated by the aspherical lens, and then passes through the linear polarizer and broadband half-wave plate in sequence to generate linearly polarized light with adjustable polarization direction. The linearly polarized light is spatially filtered by the adjustable aperture and finally focused by the focusing objective into the cylindrical prism.

[0011] The variable-angle reflector arm and optical signal acquisition system include: a collecting objective lens, a filter, a first focusing lens, a first fiber optic connector, a polarizing beam splitter prism, a second focusing lens, and a second fiber optic connector; the light reflected by the cylindrical prism is collected by the collecting objective lens, passes through the filter, and enters the polarizing beam splitter prism. S-polarized light is reflected while P-polarized light passes through the polarizing beam splitter prism. The reflected light and transmitted light are focused by the first focusing lens and the second focusing lens, respectively, onto the center of a photodiode or optical fiber fixed at the first fiber optic connector and the second fiber optic connector.

[0012] The microscopic spectral acquisition and imaging system includes components such as an imaging objective, a mirror, a long-pass filter, a third focusing lens, a camera, a 1:1 beam splitter, a fourth focusing lens, and a spectrometer. The imaging objective collects Raman and fluorescence signals from the sample at the interface. The light signal is first reflected by the mirror, then the excitation light is filtered out by the long-pass filter. After that, the light signal is split into reflected and transmitted parts by the 1:1 beam splitter. The reflected light is imaged onto the image plane of the camera by the third focusing lens, while the transmitted light is focused onto the slit of the spectrometer by the fourth focusing lens for spectral acquisition.

[0013] The variable-angle incident arm and photoexcitation system and the variable-angle reflection arm and photosignal acquisition system are symmetrically distributed on both sides of the sample stage and prism mounting frame, scanning the surface plasmon resonance angle and measuring Raman scattering and fluorescence spectra at the resonance angle;

[0014] The microscopic imaging device is located at the bottom of the sample stage and prism mounting frame and is used for total internal reflection fluorescence imaging and spectral acquisition.

[0015] Furthermore, the fluorescence and Raman spectral signals acquired by the microscopic spectral acquisition and imaging system are excited by surface plasmon polaritons of the metal film, and the signals originate entirely from the sample at the surface of the metal film.

[0016] Furthermore, surface plasmon polaritons of a metal film are used to excite the fluorescence and Raman spectral signals of molecules, and the penetration depth of the surface plasmon polaritons is adjusted by the rotating variable-angle incident arm and the photoexcitation system.

[0017] Furthermore, the spectral signal is collected by the variable-angle reflective arm and optical signal acquisition system on the cylindrical prism side or by the microscopic spectral acquisition and imaging system on the air side.

[0018] Furthermore, the polarization beam splitter prism in the variable angle reflector arm and optical signal acquisition system simultaneously measures the intensity of reflected light or the Raman spectrum under two orthogonal polarization states. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of a laser coaxial adjustment and fiber coupling system.

[0021] Figure 2 This is a schematic diagram of the plasmon coupling in-situ spectroscopy and imaging testing system for the interface of the present invention.

[0022] Figure 3 This is a total internal reflection fluorescence image acquired at the critical angle excitation;

[0023] Figure 4 This is the total internal reflection fluorescence spectrum collected at the critical angle excitation;

[0024] Figure 5 The graph shows the SPR reflectance spectrum of the silver film. The horizontal axis represents angle, and the vertical axis represents normalized light intensity.

[0025] Figure 6 The Raman spectrum of the 4-MBA probe molecule was collected at the SPR resonance angle;

[0026] The system comprises: 1. Frame and rotating arm drive; 2. Variable angle incident arm and photoexcitation system, including: 2-1 fiber optic flange, 2-2 aspherical lens, 2-3 linear polarizer, 2-4 half-wave plate, 2-5 adjustable aperture, 2-6 focusing objective; 3. Variable angle reflecting arm and optical signal acquisition system, including: 3-1 collecting objective, 3-2 filter, 3-3 polarizing beam splitter prism, 3-4 first focusing lens, 3-5 first fiber optic interface, 3-6 second focusing lens, 3-7 second fiber optic interface; 4. Sample stage and prism mounting bracket; 5. Microscopic spectral acquisition and imaging system, including components: 5-1 imaging objective, 5-2 reflecting mirror, 5-3 long-pass filter, 5-4 third focusing lens, 5-5 camera, 5-6 1:1 beam splitter, 5-7 fourth focusing lens, 5-8 spectrometer. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0028] In the accompanying drawings of specific embodiments of the present invention, in order to better and more clearly describe the working principle of each component in the system and show the connection relationship of each part in the device, only the relative positional relationship between each component is clearly distinguished. It does not constitute a limitation on the signal transmission direction, connection sequence, or size, dimension, and shape of each part within the component or structure.

[0029] The specific structure of the device of the present invention mainly includes, as follows: Figure 1 The laser coaxial adjustment and fiber coupling system shown, and as Figure 2 The schematic diagram of the plasmon-coupled in-situ spectroscopy and imaging test system shown includes: 1. Frame and rotating arm drive device; 2. Variable angle incident arm and photoexcitation system; 3. Variable angle reflecting arm and light signal acquisition system; 4. Sample stage and prism mounting bracket; 5. Microscopic spectroscopy acquisition and imaging system.

[0030] Laser coaxial adjustment and fiber coupling system such as Figure 1 As shown, a group of lasers with different wavelengths are combined into a single beam using a long-pass dichroic mirror. After reflection by a mirror, the beam is coupled into an optical fiber by a fiber coupler and finally transmitted into the optical path of the incident arm. The laser wavelength used in this invention can be 375nm, 405nm, 473nm, 532nm, 633nm, etc. The specific wavelength of the laser can be changed as needed.

[0031] For example Figure 2As shown, the optical excitation system and optical signal acquisition system are fixed on the variable-angle incident arm and variable-angle reflecting arm of the frame and rotating arm drive device, respectively. They can be moved along the direction of the vertical rotating arm by a displacement stage to achieve collimation adjustment of the initial optical path. The bottom microscopic imaging and spectral acquisition device is fixed on a horizontal slide rail, and its entirety can move independently relative to the rotating stage in the XY plane, thereby achieving the overlap of the collection range and the excitation region.

[0032] The specific working principle of this invention is as follows: Figure 1 As shown, laser beams emitted by a group of lasers with different wavelengths are combined by a dichroic mirror and then coupled into an optical fiber. The output end of the optical fiber is connected to... Figure 2 On the fiber flange 2-1 of the optical excitation system shown, the light beam coupled from the fiber to free space is first collimated by the aspherical lens 2-2, and then sequentially passes through the linear polarizer 2-3 and the broadband half-wave plate 2-4 to generate linearly polarized light with adjustable polarization direction. The linearly polarized light is spatially filtered by the adjustable aperture 2-5, and finally focused by the focusing objective lens 2-6 into the cylindrical prism whose center coincides with the rotation axis (focusing is not required when the laser power density is sufficient, in which case 2-6 can be removed).

[0033] After the laser undergoes total internal reflection on the lower surface of the cylindrical prism fixed at the sample stage and prism mounting bracket 4, a surface electromagnetic mode called the evanescent field is formed. This surface electromagnetic wave penetrates the metal film and excites surface plasmon polaritons at the interface formed by the metal film and air. Subsequently, the detection optical path of the light signal is split into two paths:

[0034] One of them is fixed at Figure 1 The variable-angle reflector arm and optical signal acquisition system 3 on the right are used to collect reflected and Raman scattered light. The optical signal is first collected by the collecting objective lens 3-1, then passes through the detachable filter 3-2 and enters the polarization beam splitter prism 3-3. S-polarized light is reflected by the polarization beam splitter prism 3-3, while P-polarized light passes through the polarization beam splitter prism. The reflected and transmitted light are focused by the first focusing lens 3-4 and the second focusing lens 3-6 onto the center of the photodetector or optical fiber fixed at the first optical fiber interface 3-5 and the second optical fiber interface 3-7, respectively. When the system is used to collect the intensity of reflected light, a photodetector is installed at the optical fiber interface, and the current signal is transmitted to the computer after passing through a transimpedance amplifier, a low-pass filter, and a data acquisition card. When the device is operating in Raman spectroscopy acquisition mode, an optical fiber is installed at the collecting end, and the Raman scattered signal is coupled into the spectrometer 5-8 through the optical fiber.

[0035] Another path is on the air side below the prism, where the fluorescence of the interface sample is collected through the imaging objective 5-1. The fluorescence signal is first reflected by the mirror 5-2, and then the excitation light is filtered out by the long-pass filter 5-3. After that, the fluorescence signal is divided into two parts, reflection and transmission, by the 1:1 beam splitter 5-6. The reflected light is imaged onto the image plane of the camera 5-5 through the third focusing lens 5-4, while the transmitted light is focused by the fourth focusing lens 5-7 onto the slit of the spectrometer 5-8 for spectral acquisition.

[0036] In Example 1, the fluorescence spectrum and imaging of silica fluorescent microspheres encapsulated with RBITC (Rhodamine B isocyanate) were observed.

[0037] The laser wavelength used in this embodiment is 532 nm (selected based on the excitation spectral characteristics of the fluorophore).

[0038] An ethanol solution containing silica microspheres coated with the fluorescent dye RBITC was spin-coated onto a 10mm*10mm*1mm BK7 glass substrate with a sputtered 45nm silver film. After spin-coating, the sample was assembled onto the bottom of a cylindrical prism, and the refractive indices of the substrate and the cylindrical prism were matched by cedar oil.

[0039] Connect the photodetector to the receiver of the variable-angle reflector arm signal acquisition system, such as to the polarization beam splitter prism 3-5, fiber optic coupler, or photodiode 3-7. Scan the variable-angle incident arm and photoexcitation system 2 and the variable-angle reflector arm and optical signal acquisition system 3 in opposite directions at the same speed, ensuring that the direction of the reflector arm remains at the reflection angle. When a minimum value appears in the reflection intensity curve, it indicates that surface plasmon resonance has been achieved. At this time, fluorescence imaging is performed through the optical path at the bottom of the prism to obtain... Figure 3 At the same time, fluorescence spectra were collected to obtain Figure 4 . Figure 3 The bright part is a silica microsphere containing fluorescent dye. Because the fluorescent dye is excited by the evanescent field confined to the surface of the silver film in the experiment, the image shows good contrast and no interference from the fluorescent background. Figure 4 The peak at 560-600 nm shown in the mid-fluorescence spectrum is the fluorescence emission peak of the dye RBITC.

[0040] Example 2: Surface plasmon resonance enhanced Raman scattering spectrum of silver film surface modified with 4-mercaptobenzoic acid.

[0041] The laser wavelength used in this embodiment is 532nm, and the optical power density is 10mW / cm². 2 .

[0042] The Raman spectral signal acquired in this embodiment comes from the Raman probe molecule 4-mercaptobenzoic acid.

[0043] First, a 5nm thick chromium layer was magnetron sputtered onto a 10mm*10mm*1mm BK7 glass substrate to enhance the adhesion of the silver film to the substrate. Then, a 50nm thick silver layer was magnetron sputtered onto the chromium layer surface. The resulting BK7 / chromium / silver substrate was immersed in a 10µM / L ethanol solution of 4-mercaptobenzoic acid to modify the silver film surface with the Raman probe molecule 4-mercaptobenzoic acid. Finally, the resulting BK7 / chromium / silver / 4-MBA was bonded to a cylindrical prism, with cedarwood oil applied between the substrate and the prism to match the refractive index.

[0044] Connect the photodetector to the receiver of the variable-angle reflector arm signal acquisition system, such as to the polarization beam splitter prism 3-5, fiber optic coupler, or photodiode 3-7. Use computer control software to drive the variable-angle incident arm to rotate to change the laser incident angle. Simultaneously, the variable-angle reflector arm rotates with the incident arm, and records the intensity of the reflected light at the reflection angle, thus obtaining... Figure 5 The SPR reflectance spectrum shown shows that the reflectance reaches its minimum at 44.3 degrees. At this point, the energy of the incident light is coupled into the surface plasmon polaritons of the silver film. This electromagnetic field mode, which is confined to the surface of the silver film, can be further scattered to the far field by molecules and detected.

[0045] Scan the variable-angle incident arm to the SPR resonance angle, replace the photodetector on either the first fiber optic interface 3-5 or the second fiber optic interface 3-7 with an optical fiber, and connect the fiber optic output to the spectrometer. Acquiring Raman spectra will yield results such as... Figure 6 The results show that the peaks at Raman shifts of 1150 and 1600 wavenumbers are characteristic peaks of the Raman probe molecule 4-mercaptobenzoic acid.

[0046] In the above embodiments, implementation can be achieved entirely or partially through software, hardware, firmware, or any combination thereof. When implemented using software, it can be implemented entirely or partially in the form of a computer program product. The computer program product includes one or more computer instructions. When the computer program instructions are loaded and executed on a computer, all or part of the processes or functions described in the embodiments of this application are generated. The computer can be a general-purpose computer, a special-purpose computer, a computer network, or other programmable device. The computer instructions can be stored in a computer-readable storage medium or transmitted through the computer-readable storage medium. The computer-readable storage medium can be any available medium that a computer can access or a data storage device such as a server or data center that integrates one or more available media. The available medium can be a magnetic medium (e.g., floppy disk, hard disk, magnetic tape), an optical medium (e.g., DVD), or a semiconductor medium (e.g., solid-state disk (SSD)).

[0047] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A plasmon-coupled table interfacial in-situ spectroscopy and imaging test system, characterized in that, The system comprises a laser coaxial adjustment and fiber coupling system, a rack and rotating arm driving device, a variable angle incident arm and light excitation system, a variable angle reflection arm and light signal collection system, a sample stage and prism mounting rack, and a microscopic spectrum collection and imaging system; the sample stage and prism mounting rack is provided with a cylindrical prism; The laser coaxial adjustment and fiber coupling system comprises a long-pass dichroic mirror, a set of lasers of different wavelengths, a fiber coupler, an optical fiber and a mirror; the long-pass dichroic mirror combines the set of lasers of different wavelengths into one beam, the beam is reflected by the mirror and coupled into the optical fiber by the fiber coupler, and finally transmitted to the variable angle incident arm and light excitation system; The variable angle incident arm and light excitation system comprises a fiber flange, an aspheric lens, a linear polarizer, a half-wave plate, an adjustable diaphragm and a focusing objective; the light beam coupled from the optical fiber to free space is first collimated by the aspheric lens, then sequentially passes through the linear polarizer and the broadband half-wave plate to generate linearly polarized light with adjustable polarization direction, the linearly polarized light is spatially filtered by the adjustable diaphragm, and finally focused into the cylindrical prism by the focusing objective; The variable angle reflection arm and light signal collection system comprises a collection objective, a filter, a first focusing lens, a first fiber joint, a polarization beam splitter prism, a second focusing lens and a second fiber joint; the light reflected by the cylindrical prism is collected by the collection objective, passes through the filter and enters the polarization beam splitter prism, the S-polarized light is reflected while the P-polarized light is transmitted through the polarization beam splitter prism, and the reflected light and the transmitted light are focused to the centers of photodiodes or optical fibers fixed at the first fiber joint and the second fiber joint by the first focusing lens and the second focusing lens respectively; The microscopic spectrum collection and imaging system comprises an imaging objective, a mirror, a long-pass filter, a third focusing lens, a camera, a 1:1 beam splitter, a fourth focusing lens and a spectrometer; the imaging objective collects the Raman and fluorescence signals of the sample at the interface, the light signal is first reflected by the mirror, then filtered by the long-pass filter to remove the excitation light, then divided into reflected and transmitted parts by the 1:1 beam splitter, the reflected light is imaged onto the image plane of the camera by the third focusing lens, and the transmitted light is focused onto the slit of the spectrometer by the fourth focusing lens for spectrum collection; The variable angle incident arm and light excitation system and the variable angle reflection arm and light signal collection system are symmetrically distributed on the two sides of the sample stage and prism mounting rack, the surface plasmon resonance angle is scanned, and the Raman scattering and fluorescence spectrum are measured at the resonance angle; A microscopic imaging device is arranged at the bottom of the sample stage and prism mounting rack, and is used for total internal reflection fluorescence imaging and spectrum collection.

2. The plasmon-coupled ex situ spectroscopic and imaging test system according to claim 1, characterized in that The fluorescence and Raman spectrum signals collected by the microscopic spectrum collection and imaging system are excited by the surface plasmon polariton of the metal film, and the signals completely come from the sample at the interface of the metal film.

3. The plasmon-coupled table interfacial in situ spectroscopy and imaging test system according to claim 2, wherein, The surface plasmon polariton of the metal film is used to excite the fluorescence and Raman spectrum signals of the molecules, and the penetration depth of the surface plasmon polariton is adjusted by the rotating variable angle incident arm and light excitation system.

4. The plasmon-coupled table interfacial in situ spectroscopy and imaging test system according to claim 1, wherein, The spectral signal is collected by the variable angle reflecting arm and light signal collection system on the side of the cylindrical prism or the microscopic spectrum collection and imaging system on the air side.

5. The plasmon-coupled table interfacial in situ spectroscopy and imaging test system according to claim 1, wherein, The polarization beam splitter prism in the variable angle reflecting arm and light signal collection system simultaneously measures the reflected light intensity or Raman spectrum in two orthogonal polarization states.

Images