SPR (Surface Plasmon Resonance) phase imaging system and method based on four polarization filter arrays

Abstract

The invention discloses an SPR phase imaging system based on a four-polarization filtering array, and the system comprises an incident light path which is used for generating area array laser; the SPR module is used for receiving the area array laser and carrying out SPR sensing to obtain an area array laser modulation result related to a to-be-detected substance; and the imaging light path is used for carrying out amplification and filtering modulation on the area array laser modulation result so as to carry out imaging on a four-polarization array, and carrying out matched filtering and double-differential phase resolving on a four-polarization image so as to obtain an imaging result. According to the invention, through polarization virtual image generation and cross-channel block matching, and in combination with four-dimensional collaborative filtering, the noise suppression efficiency is improved; and through double differential phase resolving, the dynamic range is expanded to 0.06 RIU, and the resolution ratio is superior to that of the RIU. The invention solves the problem of mutual restriction of dynamic range and resolution in the traditional SPR technology, and is suitable for high-sensitivity biomolecule interaction detection.

Description

Technical Field

[0001] This invention relates to the field of optical sensing technology, specifically to a surface plasmon resonance (SPR) phase imaging system and method based on a four-polarization filter array (PFA), which is suitable for high-resolution, wide dynamic range detection of biomolecular interactions, cell membrane dynamics research, and high-sensitivity biochemical sensing. Background Technology

[0002] Surface plasmon resonance (SPR) technology is widely used in the analysis of biomolecular interactions due to its advantages of being label-free and enabling real-time detection. Traditional phase-sensitive SPR systems face the challenge of a trade-off between dynamic range and resolution: insufficient noise suppression during wide-range measurements, while high-resolution modes often sacrifice detection range. Although existing high-resolution instruments have achieved resolutions that are... The resolution is RIU, but its detection range is less than 0.01 RIU, while the resolution of instruments with a large detection range can only reach RIU. RIU range. Existing noise reduction methods (such as temporal smoothing or neural networks) struggle to balance real-time performance and accuracy, and the application of polarization cameras in SPR imaging has not yet fully optimized noise suppression strategies. Therefore, there is an urgent need for a method that can simultaneously achieve a wide dynamic range (>0.06 RIU) and high resolution (…). RIU's SPR detection solution. Summary of the Invention

[0003] The purpose of this invention is to address the problems of mutual constraints between dynamic range and resolution, and insufficient noise suppression in existing SPR phase detection technologies, and to propose an SPR phase imaging system and method based on a four-polarization filter array. This invention can achieve a wide dynamic range (>0.06 RIU) and high resolution (…). RIU (Reactive Ingredients and Unsuppliers) synergistic detection provides a precise measurement tool for biomolecular interaction analysis and biochemical sensing.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The first aspect of this invention provides an SPR phase imaging system based on a four-polarization filter array, comprising:

[0006] Incident light path, used to generate area laser array;

[0007] The SPR module is used to receive the area laser and perform SPR sensing to obtain the area laser modulation result related to the substance under test.

[0008] The imaging optical path is used to amplify and filter the modulation result of the area array laser to image on a four-polarization array, and to perform matched filtering and double differential phase calculation on the four-polarization image to obtain the imaging result.

[0009] In some embodiments, the incident optical path includes a line laser source, a collimating lens, a polarizing lens, a beam expander, and a pupil arranged sequentially along the same optical axis.

[0010] In some embodiments, the SPR module includes a prism, a metal thin film formed on the surface of the prism, and a detection fluid chip disposed on one side of the metal thin film.

[0011] In some embodiments, the metal thin film includes an adhesion layer and an SPR active layer sequentially deposited on the prism surface facing the detection fluid chip side by electron beam evaporation.

[0012] In some embodiments, the imaging optical path includes an objective lens, a half-wave plate, and a four-polarization CMOS image sensor arranged sequentially along the optical axis.

[0013] In some embodiments, the angle between the fast axis of the half-wave plate and the s-polarization direction is 22.5° ± 1°; and / or

[0014] The mechanical adjustment accuracy of the half-wave plate should be better than 0.5°.

[0015] In some embodiments, the matched filtering and dual-difference phase resolution of the four-polarization image includes:

[0016] A virtual polarization measurement image is constructed based on the light intensity conservation relationship of the four polarization channels:

[0017] =

[0018] in, , , , To capture four-channel polarization images in real time; , , , Four sets of virtual polarization measurement images;

[0019] Construct a four-dimensional data block containing the current frame polarization image, the current frame virtual polarization measurement image, the previous frame polarization image, and the previous frame virtual polarization measurement image;

[0020] Using an image of any polarization direction among the four polarization directions as a reference, similar block matching is performed within a unit pixel block and a set search window range, and the matching results are shared with other polarization components.

[0021] The matched four-dimensional data block is subjected to linear transformations in spatial dimension, time dimension and polarization dimension in sequence to obtain the transformation result; the transformation result is then subjected to hard threshold filtering and then linear inverse transformations in polarization dimension, time dimension and spatial dimension in sequence.

[0022] The imaging result is obtained by performing double-difference interferometric phase calculation on the inverse transformation result.

[0023] In some embodiments, the formula used for the double-difference interferometric phase solution is as follows:

[0024]

[0025] Where α is the polarization angle of the incident light; and These are the field intensity coefficients for s-polarized light and p-polarized light, respectively. and These are the phases of s-polarized light and p-polarized light, respectively; , where is the phase difference between s-polarized light and p-polarized light; Total light intensity.

[0026] In some embodiments, the method further includes performing the linear inverse transform as follows:

[0027] The results of the linear inverse transform are optimized using Wiener filtering.

[0028] A second aspect of the present invention provides an SPR phase imaging method based on a four-polarization filter array, comprising:

[0029] Generates area laser array;

[0030] The area array laser is received and SPR sensing is performed to obtain the area array laser modulation result related to the substance under test;

[0031] The area array laser modulation result is amplified and filtered to form an image on a four-polarization array. The four-polarization image is then subjected to matched filtering and double-difference phase calculation to obtain the imaging result.

[0032] This invention has the following characteristics and beneficial effects:

[0033] This invention improves noise suppression efficiency by generating polarization virtual images and cross-channel block matching, combined with four-dimensional collaborative filtering; and expands the dynamic range and improves resolution through dual-differential phase resolution. This invention solves the problem of the trade-off between dynamic range and resolution in traditional SPR technology, and is suitable for high-sensitivity detection of biomolecular interactions. Specifically:

[0034] 1. Collaborative Innovation of Optics and Algorithms:

[0035] Through the physical constraints of the four polarization array ( This generates virtual channels to improve data redundancy.

[0036] By combining spatial, temporal, and polarization three-dimensional information, the noise suppression efficiency is significantly improved compared to BM4D (Block-Matching 4D filtering).

[0037] 2. Performance Breakthrough:

[0038] The dynamic range is extended to 0.06 RIU (0.036 RIU using traditional methods), and the resolution is better than... RIU;

[0039] Real-time processing speed of 10Hz, supporting 1nM protein binding kinetics detection (KD= M). Attached Figure Description

[0040] Figure 1 This is a schematic diagram of an SPR phase imaging system based on a four-polarization filter array according to an embodiment of the present invention.

[0041] Figure 2 for Figure 1 The diagram shows the micro-polarization array arrangement of the four-polarization CMOS image sensor in the imaging system shown.

[0042] Figure 3 for Figure 1 The diagram shows the modulation and filtering of the light field by pixels with four polarization components in the imaging system shown.

[0043] Figure 4 for Figure 1 The flowchart shows the PPBM4D noise reduction method used in the data processing module of the imaging system.

[0044] Figure 5 (a) and (b) are Figure 1 The image shows a comparison of the noise reduction effects of PPBM4D used in the data processing module of the imaging system.

[0045] Figure 6 Figures (a) and (b) are dynamic range and resolution verification curves of the imaging system provided in the embodiments of the present invention.

[0046] In the picture:

[0047] 100 - Incident light path, 101 - Laser, 102 - Collimating lens, 103 - Polarizing lens, 104 - Beam expander, 105 - Pupil;

[0048] 200-Imaging optical path, 201-Objective lens, 202-Half-wave plate, 203-Four-polarization CMOS image sensor, 204-Data processing module;

[0049] 300-SPR module, 301-prism, 302-metal thin film, 303-fluidic chip for detection. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this application clearer, the application will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for explaining this application and are not intended to limit this application.

[0051] Conversely, this application covers any alternatives, modifications, equivalent methods, and schemes made within the spirit and scope of this application as defined by the claims. Furthermore, to provide the public with a better understanding of this application, certain specific details are described in detail below. However, this application can be fully understood by those skilled in the art even without these detailed descriptions.

[0052] See Figure 1 The first aspect of the present invention provides an SPR phase imaging system based on a four-polarization filter array, comprising an incident optical path 100, an SPR module 300, and an imaging optical path 200; wherein,

[0053] Incident light path 100 is used to generate area array laser;

[0054] The SPR module 300 is used to receive the area array laser and perform SPR sensing to obtain the area array laser modulation result related to the substance to be tested.

[0055] Imaging optical path 200 is used to amplify and filter the modulation results of the array laser to image on a four-polarization array, and to perform matched filtering and double differential phase calculation on the four-polarization image to obtain the imaging result.

[0056] In some embodiments, in this phase imaging system, the optical axis direction is defined as the z-axis, and the plane perpendicular to the optical axis is defined as the xy-plane, thereby forming an xyz coordinate system. Figure 1 G1 and G2 in the diagram represent the light propagation directions of the incident light path 100 and the imaging light path 200, respectively.

[0057] In some embodiments, the incident optical path 100 includes a laser 101, a collimating lens 102, a polarizing lens 103, a beam expander 104, and a pupil 105 arranged sequentially along a common first optical axis.

[0058] In one specific embodiment of this application, the laser 101 is a He-Ne laser with a wavelength of 632.8nm (product model: Thorlabs HNL050L-EC), with an output power of 5mW, and the diameter of the generated line laser beam is 1.5mm. The line laser beam is collimated sequentially by a collimating lens 102, a polarizing lens 103 (considering that the degree of polarization affects the phase calculation of the subsequent imaging optical path, in order to avoid the mixing of different polarization components in channels with other polarization angles, the degree of polarization of the polarizing lens 103 should be greater than 20:1) and a 5× beam expander 104 (beam expander model: Daheng Optoelectronics GCO-2503, the beam expansion ratio is adjustable from 5 to 10 times, and 5 times is used in this embodiment). After being collimated by the pupil 105, the beam spot diameter is constrained to 8mm to ensure that the incident light uniformly covers the sensing area in the SPR module 300.

[0059] In some embodiments, the SPR module 300 is implemented using a Kretschmann prism assembly, which includes a prism 301, a metal thin film 302 formed on the surface of the prism 301, and a detection fluid chip 303 disposed on one side of the metal thin film 302. During detection, the fluid to be tested enters the SPR module 300 through the detection fluid chip 303, flows to the side of the metal thin film 302 closest to the detection fluid chip 303, and thus enters the detection range of the SPR module 300.

[0060] In one specific embodiment of this application, the prism 301 is made of ZF5 glass, with a refractive index n=1.734 under 632.8 nm light irradiation. The incident angle is adjusted within the range of 60°~75° to optimize SPR excitation conditions. The metal thin film 302 includes an adhesion layer and an SPR active layer sequentially deposited on the surface of the prism 301 facing the detection fluid chip 303 using electron beam evaporation (achieved using a Temescal FC-2000 electron beam evaporation coating system). In this embodiment, the adhesion layer is 3 nm of Cr with a deposition rate of 0.2 Å / s, and the SPR active layer is 30 nm of Au with a deposition rate of 1 Å / s. The surface roughness of the metal thin film 302 is <1 nm RMS, which can be verified using AFM. The detection fluid chip 303 is used for introducing the fluid to be tested.

[0061] In some embodiments, the imaging optical path 200 includes a polarization modulation module and a data processing module 204.

[0062] The polarization modulation module includes an objective lens 201, a half-wave plate 202, and a four-polarization CMOS image sensor 203 arranged sequentially along a common second optical axis. The objective lens 201 is used to magnify and focus the image to achieve higher spatial resolution imaging. The half-wave plate 202 is used to modulate the p-polarization component and the s-polarization component separately to obtain suitable polarization state components, and finally, a four-polarization image is obtained on the four-polarization CMOS image sensor 203. , , , The corresponding light intensity relationship.

[0063] In one specific embodiment of this application, the objective lens 201 is a telecentric imaging lens (integrated type) of Daheng Optoelectronics GCO-232 or a Promis long working distance APO objective lens (×10). The fast axis of the half-wave plate 202 has an angle of 22.5°±1° with the s-polarization direction. It is an achromatic half-wave plate of model Thorlabs AHWP05M-600 with a MgF2 substrate and a retardation of λ / 2 @630±10nm. The half-wave plate 202 is mounted on a rotating adjustment frame (not shown in the figure). The mechanical adjustment accuracy should be better than 0.5°. In this embodiment, the adjustment accuracy is ±0.1° to ensure optimization of the polarization state of the incident light. The quad-polarization CMOS image sensor 203 uses a Sony IMX250MZR global shutter, employing monochrome mode with an exposure time of 1ms and outputting via a 16-bit ADC. The pixel polarization array of the CMOS image sensor 203 uses a 4×4 pixel period (4 pixels each at 0°, 45°, 90°, and 135°), with a single pixel size of 3.45μm×3.45μm and a polarization extinction ratio >100:1 @633nm. Figure 2 As shown.

[0064] The basic principle of the modulation of SPR reflected light by the quad-polarization CMOS image sensor 203 is that each pixel in each polarization direction is only allowed to pass through the light field of that polarization component, such as... Only the y-direction polarization component is allowed to pass through, such as Figure 3 As shown in the figure, the modulation and filtering of the light field by the pixels with four polarization components are given respectively. E(x) and E(y) in the coordinate axis represent the light field components in the x and y directions in the plane perpendicular to the light propagation direction, respectively.

[0065] Data processing module 204 is used to process the four-polarization images captured by four-polarization CMOS image sensor 203. Matched filtering and dual-differential phase resolution are performed to obtain the final imaging result. The method used by data processing module 204 will be referred to as the "PPBM4D noise reduction method" below; for details, please refer to [link to documentation]. Figure 4 This includes the following steps:

[0066] Step 1: Matched Filtering

[0067] Step 1-1: Generation of Virtual Polarization Measurement Image

[0068] Based on the light intensity conservation relationship of the four polarization channels, redundant data is constructed to generate four sets of virtual polarization measurement images (e.g., ):

[0069] =

[0070] in, , , , To capture four-channel polarization images in real time, a PC-based acquisition program written in Python can be used for real-time acquisition. , , , Four sets of virtual polarization measurement images;

[0071] Construct a four-dimensional data block containing the current frame polarization image, the current frame virtual polarization measurement image, the previous frame polarization image, and the previous frame virtual polarization measurement image (four-dimensional refers to the spatial dimension of x and y, the time dimension, and the polarization dimension).

[0072] Steps 1-2: Reference Channel Block Matching

[0073] Using an image of any polarization direction among the four polarization directions as a reference, similar block matching is performed within a unit pixel block and a set search window range, and the matching results are shared with other polarization components to reduce redundant calculations and improve the algorithm's running efficiency.

[0074] In one specific embodiment of this application, a polarization image is used. Based on this, the size of the unit pixel block is 8×8 pixels, the search window range is 40×40 pixels, and a grid scanning method is used for similar block matching, and the matching results are shared with ;

[0075] Steps 1-3, Four-dimensional Collaborative Filtering

[0076] First, the matched four-dimensional data blocks are subjected to linear transformations in spatial dimension, time dimension and polarization dimension in sequence to obtain the transformation results;

[0077] In one specific embodiment of this application, a two-dimensional Haar wavelet transform or a two-dimensional Bior 1.5 wavelet transform is used for the spatial dimension, a 4×4 one-dimensional Hadamard transform is used for the time dimension, and an 8×8 one-dimensional discrete cosine transform (DCT) is used for the polarization dimension.

[0078] Subsequently, hard thresholding is applied to the transformation results: a fixed threshold is used for hard thresholding, that is, noise components with an absolute value < 3σ in the transformation results are discarded, and the noise standard deviation is estimated by using the non-patterned regions of the image.

[0079] Next, inverse transformation reconstruction is performed: the inverse transformation of the above spatial dimension, polarization dimension, and frequency domain dimension transformation process is performed to return to the initial video signal dimension.

[0080] Furthermore, step 1, following steps 1-3, also includes the following steps:

[0081] Steps 1-4: Wiener Filter Optimization

[0082] A standard Wiener filter (with a regularization coefficient of 1) is used to perform a secondary filter on the video dimension image signal obtained from the inverse transform reconstruction in steps 1-3, thereby achieving a better noise suppression effect. Specifically, this includes:

[0083] First, re-block matching is performed, based on the video dimension image signal obtained by inverse transform reconstruction;

[0084] Next, perform the same four-dimensional transformation as in steps 1-3:

[0085] Spatial dimension: Haar wavelet transform

[0086] Time dimension: 4×4 Hadamard transform

[0087] Polarization dimension: 8×8 DCT transform

[0088] Next, Wiener filtering is performed, and the standard Wiener filtering method is used to process the signal in the transform domain.

[0089] Finally, the inverse transform returns to the video domain.

[0090] Step 2, Phase Calculation

[0091] The inverse transform result is subjected to double-difference interferometric phase calculation to obtain the imaging result. The formula used for double-difference interferometric phase calculation is as follows:

[0092]

[0093] Where α is the incident light polarization angle, set to 45°±2°; and These are the field intensity coefficients for s-polarized light and p-polarized light, respectively. and These are the phases of s-polarized light and p-polarized light, respectively; , where is the phase difference between s-polarized light and p-polarized light; Total light intensity.

[0094] Figure 5 A schematic diagram illustrating the effect of a PPBM4D noise reduction method provided by an embodiment of the present invention is given. Figure 5 Image (a) shows the phase-calculated signal obtained by introducing steady-state deionized water into the SPR module 300 before applying the PPBM4D denoising method. The fluctuations indicate measurement noise, with a noise standard deviation of 5.82e-5 rad. The PPBM4D denoising method of this embodiment is applied to this image data, and the phase-calculated signal is redrawn as shown below. Figure 5 In (b), it can be observed that the noise fluctuation is reduced and the noise standard deviation is reduced to 2.74e-5 rad;

[0095] Figure 6 The dynamic range and resolution verification curves of the embodiments of the present invention are given. Figure 6 Figure (a) shows the results of the dynamic range calibration, with the phase-refractive index curves calibrated using a standard glycerol solution gradient (mass fraction 0%–44%, corresponding to 1.333–1.393 RIU). See [reference needed]. Figure 6 In (b), the linear correlation coefficient R² > 0.99.

[0096] Understandably, this invention solves the problems of mutual constraints between dynamic range and resolution, and insufficient noise suppression in existing SPR phase detection technologies. It utilizes an SPR phase imaging system and method based on a four-polarization filter array, achieving improved noise suppression efficiency through polarization virtual image generation and cross-channel block matching, combined with four-dimensional collaborative filtering. Furthermore, it expands the dynamic range through dual-differential phase calculation. Ultimately, this invention can achieve both a wide dynamic range (>0.06 RIU) and high resolution (…). The RIU (Radio-Induced Unstable) co-detection provides a precise measurement tool for biomolecular interaction analysis and biochemical sensing. Meanwhile, the acquisition mode based on a four-polarization filter array overcomes the image alignment challenge in traditional dual-CCD SPR acquisition methods, improving image consistency.

[0097] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this disclosure. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0098] Although embodiments of this disclosure have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of this disclosure, the scope of which is defined by the claims and their equivalents.

Claims

1. An SPR phase imaging system based on a four-polarization filter array, characterized in that, include: Incident light path, used to generate area laser array; The SPR module is used to receive the area laser and perform SPR sensing to obtain the area laser modulation result related to the substance under test. The imaging optical path is used to amplify and filter the modulation result of the area array laser to image on a four-polarization array, and to perform matched filtering and double differential phase calculation on the four-polarization image to obtain the imaging result.

2. The SPR phase imaging system according to claim 1, characterized in that, The incident optical path includes a line laser source, a collimating lens, a polarizing lens, a beam expander, and a pupil arranged sequentially along the optical axis.

3. The SPR phase imaging system according to claim 1, characterized in that, The SPR module includes a prism, a metal thin film formed on the surface of the prism, and a detection fluid chip disposed on one side of the metal thin film.

4. The SPR phase imaging system according to claim 3, characterized in that, The metal film includes an adhesion layer and an SPR active layer sequentially deposited on the prism surface facing the fluid chip for detection by electron beam evaporation.

5. The SPR phase imaging system according to claim 1, characterized in that, The imaging optical path includes an objective lens, a half-wave plate, and a four-polarization CMOS image sensor arranged sequentially along the optical axis.

6. The SPR phase imaging system according to claim 5, characterized in that, The fast axis of the half-wave plate makes an angle of 22.5° ± 1° with the s-polarization direction; and / or The mechanical adjustment accuracy of the half-wave plate should be better than 0.5°.

7. The SPR phase imaging system according to claim 1, characterized in that, The process of performing matched filtering and double-difference phase resolution on the four-polarization image includes: A virtual polarization measurement image is constructed based on the light intensity conservation relationship of the four polarization channels: = in, , , , To capture four-channel polarization images in real time; , , , Four sets of virtual polarization measurement images; Construct a four-dimensional data block containing the current frame polarization image, the current frame virtual polarization measurement image, the previous frame polarization image, and the previous frame virtual polarization measurement image; Using an image of any polarization direction among the four polarization directions as a reference, similar block matching is performed within a unit pixel block and a set search window range, and the matching results are shared with other polarization components. The matched four-dimensional data block is subjected to linear transformations in spatial dimension, time dimension and polarization dimension in sequence to obtain the transformation result; the transformation result is then subjected to hard threshold filtering and then linear inverse transformations in polarization dimension, time dimension and spatial dimension in sequence. The imaging result is obtained by performing double-difference interferometric phase calculation on the inverse transformation result.

8. The SPR phase imaging system according to claim 7, characterized in that, The formula used for the double-difference interferometric phase solution is as follows: Where α is the polarization angle of the incident light; and These are the field intensity coefficients for s-polarized light and p-polarized light, respectively. and These are the phases of s-polarized light and p-polarized light, respectively; , where is the phase difference between s-polarized light and p-polarized light; Total light intensity.

9. The SPR phase imaging system according to claim 7, characterized in that, Following the aforementioned inverse linear transformation, the following is also included: The results of the linear inverse transform are optimized using Wiener filtering.

10. A SPR phase imaging method based on a four-polarization filter array, characterized in that, include: Generates area laser array; The area array laser is received and SPR sensing is performed to obtain the area array laser modulation result related to the substance under test; The area array laser modulation result is amplified and filtered to form an image on a four-polarization array. The four-polarization image is then subjected to matched filtering and double-difference phase calculation to obtain the imaging result.

Images