Time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis

Abstract

The invention relates to a time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis. The time-resolved fluorescence spectrum measurement method is mainly used for accurately measuring the polarization state of sample fluorescence. According to the method, a pulse laser is used for generating a required pulse laser sequence, excitation pulses are modulated by an excitation light modulation module and then enter a fluorescence measurement module to excite a sample and collect fluorescence signals. And then, the generated fluorescence is modulated by the Stokes parameter modulation module and finally enters the detection module for fluorescence signal acquisition. By combining a time-resolved fluorescence technology and a polarization optical element, the method can accurately collect synchronous laser pulse and fluorescence electric signals, so that time-resolved Stokes parameters are calculated, and optical characteristics such as polarization degree, azimuth angle and ellipsometry are further obtained. The method can be suitable for research on anisotropic samples, chiral samples and other samples with polarization optical properties, and has wide application prospects in the fields of molecular optics, material science, life science and the like.

Description

Technical Field

[0001] The present invention relates to the field of spectroscopy, and in particular to a time-resolved fluorescence spectroscopy measurement method based on Stokes parameter analysis. Background Art

[0002] Fluorescence technology is widely used in fields such as chemistry, biology, and materials science, and plays an important role especially in analyzing the molecular structure of substances, intermolecular interactions, and biological labeling. In recent years, the fluorescence properties of chiral materials have attracted extensive attention. Chiral samples have asymmetric optical properties and have specific responses to polarized light, thereby affecting the polarization characteristics of their fluorescence signals. For example, the fluorescence polarization characteristics of chiral molecules can be used as an effective tool for molecular recognition and are widely applied in fields such as drug screening, sensor design, and biomarker analysis.

[0003] However, the existing time-resolved polarization fluorescence technology still has limitations in polarization measurement, and it is impossible to obtain complete polarization information of fluorescence, making it difficult to meet the experimental requirements for high-precision and full-parameter measurement. Traditional methods are often incomplete in characterizing the fluorescence polarization state, especially in the characterization of dynamic change processes, key information is easily lost. Therefore, how to accurately obtain the complete polarization characteristics of fluorescence on a time-resolved scale and improve the measurement accuracy to overcome the deficiencies of the existing technology has become an important problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a time-resolved fluorescence spectroscopy measurement method based on Stokes parameter analysis. Its characteristics are: while providing high-precision fluorescence lifetime measurement, it can provide comprehensive polarization fluorescence information, and important parameters such as degree of polarization, azimuth angle, ellipticity, etc. can be accurately calculated; and it has the ability to characterize polarization optical properties such as anisotropy and chirality.

[0005] To achieve the above object, a time-resolved fluorescence spectroscopy measurement device based on Stokes parameter analysis is built. The device includes a pulsed laser, an excitation light modulation module, a fluorescence measurement module, a Stokes parameter modulation module, and a detection module. The pulse sequence generated by the pulsed laser is modulated by the excitation light modulation module and then used to excite the sample; the fluorescence signal emitted by the sample is collected by the fluorescence measurement module; the fluorescence polarization state modulation is achieved through the Stokes parameter modulation module; and finally, the detection module records the light intensity information at different delays.

[0006] In the described measuring device, the excitation light modulation module consists of a wavelength selector, a polarization modulator, and an intensity modulator. Among them, the wavelength selector is composed of a filter, which is used to block the light of non-excitation wavelengths emitted by the laser, especially the light that coincides with the fluorescence band of the sample to be measured; the polarization modulator is composed of a polarizer, a half-wave plate, or a quarter-wave plate, which is used to form a specific type of excitation light polarization state; the intensity modulator is composed of a set of neutral density filters, which is used to adjust the laser intensity to meet the excitation requirements of different samples and prevent sample damage caused by over-excitation.

[0007] In the described measuring device, the fluorescence measurement module successively consists of a sample stage, a lens, and a fluorescence filter. Among them, the sample stage is used to place the sample to be measured; the lens is placed behind the sample and is perpendicular to the excitation light beam, which is used to collect and converge the fluorescence signal emitted by the sample; the fluorescence filter is used to block the scattered excitation light and prevent the scattered excitation light from entering the detector.

[0008] In the described measuring device, the Stokes parameter modulation module consists of a quarter-wave plate and an analyzer, which are the key components for realizing the measurement of Stokes parameters. By adopting the quarter-wave plate rotation method, the fast axis direction of the quarter-wave plate is rotated to change the polarization characteristics of the emitted fluorescence. The analyzer keeps the polarization direction parallel to the horizontal direction, which is used to select the light of a specific polarization direction and extract the fluorescence intensity information related to polarization, so as to complete the measurement of the Stokes parameters of fluorescence.

[0009] In the described measuring device, the detection module consists of a detector and a TCSPC card, which is used to record the change of fluorescence emission intensity over time, so as to realize time-resolved fluorescence measurement.

[0010] The principle of the above time-resolved fluorescence spectroscopy measuring device based on Stokes parameter analysis is as follows. The Stokes parameters consist of four parameters and can be expressed as

[0011]

[0012] where I x , I y , I +45 , I -45 , I R , I L represent the light intensities of the horizontal direction, the vertical direction, the direction with an angle of +45° with the horizontal direction, the direction with an angle of -45° with the horizontal direction, the right circularly polarized light, and the left circularly polarized light respectively. The Stokes parameters have the following physical meanings: S0 represents the sum of the intensities of the horizontal and vertical components, S1 represents the difference in intensities between the horizontal and vertical components, S2 represents the difference in intensities between the +45° and -45° components, and S3 represents the difference in intensities between the right-handed and left-handed components.

[0013] For a linear polarizer with a horizontal polarization direction, its Mueller matrix is

[0014]

[0015] For a quarter-wave plate with the fast axis direction making an angle θ with the horizontal direction, its Mueller matrix is

[0016]

[0017] The Stokes parameters of the fluorescence emitted by the sample and the Stokes parameters of the light transmitted through the linear polarizer have the following relationship

[0018] S out = N × M × S in

[0019] Since only the intensity (S'0) of the light transmitted through the linear polarizer can be detected during the calculation, only the first component of the Stokes parameters needs to be calculated. The relationship between the magnitude of the outgoing light intensity and the parameters is

[0020]

[0021] Therefore, the general expression for the intensity of the light transmitted through the linear polarizer is

[0022]

[0023] Comparing the above two equations, we get S0 = A - C, S1 = 2C, S2 = 2D, S3 = B

[0024] Among them, the four coefficients A, B, C, and D can be obtained by Fourier analysis

[0025]

[0026]

[0027] This method requires ensuring that the fast axis direction of the quarter-wave plate should be adjusted at equal intervals within the range of 0° to 180° with respect to the horizontal direction, and at least eight groups of data should be recorded to ensure the accuracy of the measurement.

[0028] Preferably, for the above excitation light modulation module, the wavelength selector can be replaced by a simple monochromator composed of a grating and a slit to achieve flexible selection of the pulse light wavelength and bandwidth for different experimental requirements.

[0029] Preferably, for the above excitation light modulation module, the polarization modulator can adopt a Becker polarization compensator to achieve flexible modulation of the polarization state of light within a wide wavelength range.

[0030] Preferably, for the above excitation light modulation module, the intensity modulator can use a variable neutral density filter to achieve continuous adjustment of intensity.

[0031] Preferably, for the above fluorescence measurement module, a neutral density filter can be added to control the fluorescence intensity and prevent the detector from being saturated and damaged.

[0032] Preferably, for the above Stokes parameter modulation module, the quarter-wave plate should be an achromatic quarter-wave plate. Compared with a quarter-wave plate with a fixed wavelength, the achromatic quarter-wave plate has more stable phase delay characteristics in a wider spectral range, thereby reducing the polarization adjustment error caused by the fluorescence wavelength shift.

[0033] Preferably, for the above Stokes parameter modulation module, the analyzer can be a Glan-Taylor prism to achieve high-precision polarization optical measurement. The Glan-Taylor prism has a high polarization extinction ratio and stability, thus effectively improving the measurement accuracy and signal-to-noise ratio, and is suitable for situations with high precision requirements.

[0034] Optionally, the above pulsed laser can be a nanosecond, picosecond, or femtosecond laser, and the specific model is determined according to the time resolution required by the experiment.

[0035] Optionally, for the above excitation light modulation module, the polarization modulator can use a polarizer and a quarter-wave plate to form a circularly polarized light generator to achieve left / right circularly polarized light excitation.

[0036] Optionally, for the above fluorescence measurement module, the fluorescence filter can use a high-pass filter to achieve passing only the sample fluorescence and filtering out the remaining non-fluorescent signals.

[0037] Optionally, the fluorescence techniques adopted by the above detection module include time-correlated single photon counting (TCSPC), optical Kerr gate technology, streak camera measurement method, and frequency up-conversion technology. It can be selected according to different application requirements to meet the requirements of different experiments.

[0038] In summary, the time-resolved fluorescence spectroscopy measurement method based on Stokes parameter analysis provided by the present invention has significant innovation and superiority. By combining time-resolved fluorescence technology with Stokes parameter measurement, the polarization information of fluorescence in the dynamic process can be obtained more comprehensively and accurately. This method shows high sensitivity and accuracy in the research of anisotropic, chiral, or other fluorescent samples with polarization optical properties. Compared with the conventional time-resolved polarization fluorescence technology, this method can analyze the complete polarization information of the fluorescence signal, including linear polarization, circular polarization, and elliptical polarization components, making the measurement results more complete and reliable, and providing a more accurate analysis means for fields such as molecular recognition, drug screening, and material research. Description of the Drawings

[0039] Figure 1 Schematic diagram of a time-resolved fluorescence measurement system based on Stokes parameter analysis (taking TCSPC as an example). In the figure, 1 is a pulsed laser, 2 is an excitation light modulation module, 3 is a first filter, 4 is a polarizer, 5 is a first neutral density filter, 6 is a fluorescence measurement module, 7 is a sample stage, 8 is a lens, 9 is a second filter, 10 is a second neutral density filter, 11 is a Stokes parameter modulation module, 12 is a quarter-wave plate, 13 is an analyzer, 14 is a detection module, 15 is a detector, and 16 is a TCSPC card.

[0040] Figure 2 Schematic diagram of the time recording principle of the TCSPC card. From top to bottom are the laser pulse sequence (with a period of T), the photons received by the detector, and the time difference between the laser pulse signal and the fluorescence signal recorded by the TCSPC card. Specific implementation manner

[0041] The object of the present invention is to provide a time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis, which can obtain the complete polarization information of fluorescence through multiple measurements.

[0042] The present invention will be described below in conjunction with specific implementation manners. It should be understood that the drawings are only for illustrative purposes, showing only schematic diagrams, not physical diagrams, and should not be construed as a limitation of this patent. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0043] A time-resolved fluorescence spectrum measurement device based on Stokes parameter analysis, the optical path of which is as Figure 1 shown:

[0044] This system is based on the existing TCSPC fluorescence detection device, and components such as a quarter-wave plate (12) and an analyzer (13) are added to realize Stokes parameter measurement. The measurement system includes: a pulsed laser (1), an excitation light modulation module (2), including a first filter (3), a polarizer (4), a first neutral density filter (5), a fluorescence measurement module (6), including a sample stage (7), a lens (8), a second filter (9), a second neutral density filter (10), a Stokes parameter modulation module (11), including a quarter-wave plate (12), an analyzer (13), a detection module (14), including a detector (15), and a TCSPC card (16).

[0045] The pulsed laser (1) is used to generate a pulse sequence with a fixed frequency. Before exciting the sample, the pulsed laser first enters the excitation light modulation module (2), which includes a first filter (3), a polarizer (4), and a first neutral density filter (5), to block the light of unnecessary wavelengths emitted by the laser (1), change the polarization state of the laser pulse, and adjust the laser intensity. The fluorescence measurement module (6) is connected to the excitation light modulation module (2). A lens (8) is placed behind the sample stage (7) and is perpendicular to the excitation light beam, and is used to collect and converge the emitted fluorescence signal. Subsequently, the fluorescence signal successively passes through a second filter (9) to block the scattered excitation light, and passes through a second neutral density filter (10) to adjust the fluorescence intensity and prevent the signal from being too strong and causing the detector (15) to saturate. The Stokes parameter modulation module (11), which includes a quarter-wave plate (12) and an analyzer (13), is used to adjust the polarization state of the emitted fluorescence. The role of the quarter-wave plate (12) is to change the ellipticity of the fluorescence. The analyzer (13) further selectively filters the polarization direction of the fluorescence signal to obtain the polarization information in a specific direction. The detection module (14) is connected to the Stokes parameter modulation module (11) and includes a detector (15) and a TCSPC card (16). The detector (15) has a high time resolution and can accurately capture the fluorescence signal emitted by the sample and count the photon events. The TCSPC card (16) is connected to the pulsed laser (1) and the detector (15), and is used to collect the pulsed synchronous electrical signal of the laser (1) and the corresponding fluorescence electrical signal. The TCSPC card (16) generates time-resolved fluorescence data by accurately recording the time difference between the laser pulse and the fluorescence signal, thereby providing support for analyzing the change of the Stokes parameter over time.

[0046] The timing control and acquisition logic of the above time-resolved fluorescence spectroscopy measurement device for Stokes parameter analysis are as Figure 2 shown. During the time-resolved fluorescence detection process, not every laser pulse can excite detectable fluorescence photons. To optimize the data storage efficiency, the TCSPC card (16) only records the time interval between each detected fluorescence photon and the immediately following laser pulse (i.e., Figure 2 t1', t2', t3' in i ). The fluorescence lifetime can be calculated based on the time interval: t i =T - t i ', where T represents the repetition period of the laser pulse. By statistically analyzing all the recorded t i , the fluorescence lifetime curve can be finally obtained.

[0047] To make the objectives, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0048] The specific measurement steps for determining whether the sample has polarization optical properties and calculating the time-resolved fluorescence Stokes parameters are as follows:

[0049] ① Before the formal experiment, first measure the fluorescence emission spectrum and excitation spectrum of the sample to determine the excitation wavelength and detection wavelength, which serve as the basic parameters for the formal experiment.

[0050] ② Start the pulsed laser (1) for preheating. After the light source is stable, place the sample to be measured in the optical path.

[0051] ③ Adjust the polarizer (13) so that its polarization direction is horizontal, and adjust the fast axis direction of the quarter-wave plate (12) to be horizontal (this is the initial state of the experiment).

[0052] ④ Adjust the second neutral density filter (10) to an appropriate position to ensure that the fluorescence intensity remains within the effective range of the detector (15) during one full rotation of the quarter-wave plate (12).

[0053] ⑤ In each experiment, rotate the quarter-wave plate (12) counterclockwise along the direction of light propagation, and adjust the angle between the fast axis direction of the quarter-wave plate (12) and the horizontal direction at equal intervals, which are set to 0°, 22.5°, 45°, 67.5°, 90°, 112.5°, 135°, and 157.5° respectively. Record the variation of the number of photons collected over time at each angle. The data obtained in the eight experiments are denoted as N(0°,t), N(22.5°,t), N(45°,t), N(67.5°,t), N(90°,t), N(112.5°,t), N(135°,t), and N(157.5°,t) respectively.

[0054] ⑥ Under the above settings, use the TCSPC technique to record the number of fluorescence photons received at different delay times. The number of photons received per unit time is proportional to the light intensity. Therefore, the intensity information of the fluorescence signal can be indirectly obtained by counting the number of photons at different time delays. To simplify the calculation, during the experiment, factors such as the excitation light intensity and the optical path length remain unchanged. The number of photons received by the detector (15) can be used as an approximation of the light intensity for measuring the fluorescence intensity.

[0055] The general expression for the light intensity transmitted through a linear polarizer is

[0056]

[0057] where the four coefficients A, B, C, and D can be obtained by Fourier analysis

[0058]

[0059] The Stokes parameters of the fluorescence emitted by the sample have the following relationships with A, B, C, and D: S0 = A - C, S1 = 2C, S2 = 2D, S3 = B.

[0060] ⑦Based on the values of these Stokes parameters, the normalized Stokes parameters can be further obtained.

[0061] ⑧According to the definition of the Stokes parameters, theoretically, when the fluorescence is linearly polarized light, S3 = 0; when the fluorescence is circularly polarized light or elliptically polarized light, S3 ≠ 0. Therefore, in the experiment, for different delays, if the measured value of the Stokes parameter S3 is constantly zero, it can be determined that the sample does not have polarization optical properties. If the measured value of S3 is not zero, the sample has polarization optical properties.

[0062] ⑨When the normalized S3 = 1 and S1 = S2 = 0, the emitted fluorescence is right-handed circularly polarized light; when the normalized S3 = -1 and S1 = S2 = 0, the emitted fluorescence is left-handed circularly polarized light; when 0 < S3 < 1 after normalization, the emitted fluorescence is right-handed elliptically polarized light; when -1 < S3 < 0 after normalization, the emitted fluorescence is left-handed elliptically polarized light.

[0063] ⑩Polarization information such as degree of polarization (DOP), azimuth angle (α), and ellipticity (β) can be calculated by the following formulas.

[0064] The time-resolved fluorescence spectroscopy measurement method based on Stokes parameter analysis of the present invention can accurately measure and analyze the polarization fluorescence characteristics of the sample, provide accurate optical measurement data for in-depth study of the optical properties of materials, characterization of the polarization characteristics of materials, and various scientific research, and has broad application prospects.

Claims

1. A time-resolved fluorescence spectroscopy measurement method based on the analysis of Stokes parameters, characterized in that: It includes a pulsed laser, an excitation light modulation module, a fluorescence measurement module, a Stokes parameter modulation module, a detection module, and a Stokes parameter analysis algorithm.

2. The time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis according to claim 1, wherein: It includes the following measurement steps: (1) generating a pulsed laser sequence through the pulsed laser; (2) modulating the excitation pulse using the excitation light modulation module; (3) the modulated pulsed laser enters the fluorescence measurement module to excite the sample and collect the emitted fluorescence signal; (4) the collected fluorescence signal enters the Stokes parameter modulation module for polarization state modulation; (5) synchronously collecting the laser pulse signal and the fluorescence electrical signal through the detection module; (6) calculating the Stokes parameters based on the collected data to obtain the fluorescence polarization state information of the sample.

3. The time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis according to claim 1, characterized in that: By measuring the Stokes parameters of the fluorescence signal emitted by the sample, the polarization characteristics of the sample's luminescence can be analyzed, thereby determining whether it has anisotropy, chirality, or other polarization optical properties.

4. The time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis according to claim 1, wherein: The device is applicable to the fluorescence polarization measurement of samples and can provide information on the change of the fluorescence polarization state over time.

5. The time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis according to claim 1, characterized in that The described excitation light modulation module includes a wavelength selector, a polarization modulator, and an intensity modulator, and can achieve complete optical parameter modulation of the excitation pulse.

6. The time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis according to claim 1, characterized in that: The described Stokes parameter modulation module includes a quarter-wave plate and a polarizer. Adjust the fast axis direction of the quarter-wave plate and the polarization direction of the polarizer to be horizontal, fix the position of the polarizer, and rotate the quarter-wave plate in each experiment.

7. The time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis according to claim 1, wherein The described Stokes parameter analysis algorithm requires that the fast axis direction of the quarter-wave plate in the Stokes parameter modulation module should be adjusted at equal intervals within the range of 0° to 180° with respect to the horizontal direction, and at least eight groups of data should be recorded to ensure the accuracy of the measurement.

8. The time-resolved fluorescence spectrum measurement method based on Stokes parameter analysis according to claim 1, characterized in that The described Stokes parameter analysis algorithm is solved by applying the Mueller matrix, and information such as the degree of polarization, azimuth angle, and ellipticity is further calculated based on the Stokes parameters.

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