Two-Photon Fluorescence Z-Scan Device and Measurement Method

Abstract

The present invention belongs to the technical field of nonlinear optics, and particularly relates to a two-photon fluorescence Z-scan device and a measurement method. The method includes: S1: Using a translation stage to move the sample to be measured to the focal position z1 of the focusing lens, obtaining the wavelength window of the current two-photon fluorescence signal, and the fluorescence collection device correspondingly collects the two-photon fluorescence signal from the sample to be measured; the fluorescence intensity measurement module, the first energy meter, and the second energy meter send their respective collection results to the energy collection module; S2: Controlling the sample to be measured to move along the optical axis of the focusing lens at a preset step size, repeating step S1 until h groups of measurement data are obtained; S3: The computer completes the Z-scan of the sample to be measured and the measurement of the two-photon fluorescence data based on the h groups of measurement data. The present invention has the ability to measure the two-photon fluorescence intensity and the two-photon absorption coefficient under different light intensities.

Description

Technical Field

[0001] The present invention belongs to the technical field of nonlinear optics, and particularly relates to a two-photon fluorescence Z-scan device and a measurement method. Background Art

[0002] The two-photon fluorescence (TPF) process has a wide range of applications in multiple fields, including but not limited to biological imaging, drug development, materials science, high-sensitivity sensors, and molecular dynamics. For example, two-photon microscopy technology is often used for imaging living cells and tissues, capable of providing high-resolution three-dimensional images; two-photon fluorescence probe technology can be used to study the optical properties of new materials, especially nanomaterials and polymers; it is also an intuitive detection index for studying phenomena such as molecular dynamics and energy transfer. Since the light intensity of two-photon fluorescence is proportional to the light energy of two-photon absorption, both have the potential for measuring the nonlinear coefficient. Among them, as early as 2001, D. Aoulianov et al. built a nonlinear coefficient measurement method based on two-photon fluorescence. This method uses a CCD detector to image the two-photon fluorescence region of the sample and obtain the ADC count intensity, and this ADC count intensity is proportional to the square of the incident light intensity, and its slope k is proportional to the two-photon absorption coefficient. Therefore, through the calibration of the two-photon absorption coefficient of the rhodamine B solution, the two-photon absorption coefficient of the sample can be obtained.

[0003] The instruments required for the TPF method are expensive and the measurement time is long; at the same time, since a calibration solution is required for the measurement, and the calibration solution has strict requirements on preparation processes, optical path settings, and laser parameters, and in reality, the reference sample is affected by various environments, and its two-photon fluorescence quantum efficiency (proportional to the two-photon absorption coefficient) is easily affected by the environment and changes, resulting in a decrease in the final measurement accuracy.

[0004] The Chinese invention patent titled "An in-situ detection device for nonlinear optical properties with full-band adjustable polarization" (publication number: CN116879242A, publication date: October 13, 2023) realizes the integrated measurement of in-situ femtosecond Z-scan testing, second harmonic testing, and fluorescence emission excitation spectrum. The Chinese invention patent titled "A method for measuring the nonlinear optical properties of luminescent materials" (publication number: CN102879334B, publication date: June 3, 2015) uses optical filtering technology to eliminate the influence of fluorescence emission on Z-scan testing, but essentially still measures Z-scan and fluorescence radiation separately, and does not combine Z-scan with two-photon fluorescence measurement technology.

[0005] The paper "Two-photon absorption cross-section results of three tri-branched derivatives: A comparison between open-aperture Z-scan and two-photon excited fluorescence method" published in the journal Optik measured the two-photon absorption cross-sections of three DCM dyes of dicyanopyridine under the excitation of femtosecond pulses of a titanium sapphire laser based on the TPF method and the open-aperture Z-scan method. The experimental transmission curve was approximated with the corresponding model curve to obtain the two-photon absorption cross-section of the sample. However, the TPA cross-section data measured for the same substance by the direct (Z-scan) and indirect (two-photon fluorescence) methods are very different. For example, under femtosecond excitation at a wavelength of 800 nm, the TPA cross-section of rhodamine 6G measured by the fluorescence method is 134 GM, which is an order of magnitude higher than the 12.8 GM measured by the Z-scan method.

[0006] Similarly, Yaochuan Wang et al. studied the two-photon absorption cross-sections of several tri-branched derivatives using the femtosecond open-aperture Z-scan and two-photon excited fluorescence methods. The values of the two-photon absorption cross-sections measured by the two methods were compared. The results show that there are obvious differences in the absolute values of the two-photon absorption cross-sections measured by different techniques. The absorption cross-section measured by the two-photon excited fluorescence method is 2.1 - 2.3 times larger than the absorption cross-section measured by the open-aperture z-scan method. And this difference is very likely caused by the systematic errors of different measurements.

[0007] From the above description, it shows that the method of measuring the absorption cross-section by two-photon fluorescence has certain advantages in measurement sensitivity compared with the conventional Z-scan method, but there are certain differences in the measurement results of the two. Therefore, for materials with two-photon fluorescence, how to use the two-photon fluorescence spectroscopy measurement technology to improve the measurement sensitivity of the two-photon absorption cross-section is an urgent problem to be solved. Summary of the Invention

[0008] In view of this, the present invention aims to provide a two-photon fluorescence Z-scan device and measurement method to solve the problem of the high cost of measuring the two-photon absorption cross-section by the two-photon fluorescence intensity in the prior art. The present invention has the advantages of being easy to build and low cost. It not only has the ability to measure the two-photon fluorescence intensity and the two-photon absorption coefficient under different light intensities at the same time, but also can improve the measurement sensitivity of the two-photon absorption coefficient by comparing the two sets of data with each other.

[0009] To achieve the above object, the technical solution of the present invention is realized as follows:

[0010] A two-photon fluorescence Z-scan device includes a pulsed laser, a pulse generator, a fluorescence intensity measurement module, an attenuation module, a beam splitter, a first energy meter, a second energy meter, an energy collection module, a fluorescence collection module, a focusing lens, a collection lens, and a computer. Among them, the pulsed laser controls the laser pulse to delay a preset time through the pulse generator to externally trigger the fluorescence intensity measurement module, so that the fluorescence intensity measurement module receives the laser pulse and the corresponding two-photon fluorescence signal; the laser pulse emitted by the pulsed laser also enters the beam splitter through the attenuation module for beam splitting. One beam of laser is received by the first energy meter as a reference beam, and the other beam of laser is irradiated onto the sample to be measured through the focusing lens as a measurement beam. The sample to be measured generates a two-photon fluorescence signal under the excitation of the measurement beam. The fluorescence collection device collects the two-photon fluorescence signal and sends the collected two-photon fluorescence signal to the fluorescence intensity measurement module. The measurement beam passing through the sample to be measured is received by the second energy meter through the collection lens;

[0011] The sample to be measured moves along the optical axis of the focusing lens at a preset step size. The fluorescence collection device is relatively stationary with respect to the sample to be measured, and correspondingly collects the two-photon fluorescence signal from the sample to be measured, and sends the correspondingly collected two-photon fluorescence signal to the fluorescence intensity measurement module. The second energy meter uses the collection lens to correspondingly receive the measurement beam passing through the sample to be measured; the first energy meter and the second energy meter send their respective collection results to the energy collection module; the computer completes the Z-scan of the sample to be measured and the measurement of the two-photon fluorescence data according to the data obtained by the energy collection module and the fluorescence intensity measurement module respectively.

[0012] Further, the fluorescence intensity measurement module is a combination of a band-pass filter and a photodiode or a spectrometer. If the fluorescence intensity measurement module uses a combination of a band-pass filter and a photodiode, the total number of electrons measured by the photodiode is the fluorescence intensity.

[0013] Further, the attenuation module includes a diaphragm, a half-wave plate, and a beam splitting prism arranged in sequence along the optical axis. The attenuation module is used to attenuate and control the energy of the laser pulse.

[0014] Further, the fluorescence collection module is a collection device or an integrating sphere collection device. Among them, the collection device consists of a fiber collimator and a fiber connected in sequence. The fiber collimator collects the two-photon fluorescence signal generated by the sample to be measured under the excitation of the measurement beam, and the fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module;

[0015] The integrating sphere collection device includes a fiber collimator, a fiber, an integrating sphere, and a cuvette slot built in the integrating sphere. The fiber is connected to the fiber collimator. The cuvette slot is used to place the sample to be measured. The measurement beam irradiates the sample to be measured. The fiber collimator collects the two-photon fluorescence signal generated by the sample to be measured under the excitation of the measurement beam, and the fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module.

[0016] Furthermore, the two-photon fluorescence Z-scanning device further includes a translation stage for adjusting the positions of the sample to be measured and the fluorescence collection module.

[0017] A two-photon fluorescence Z-scanning measurement method implemented using the two-photon fluorescence Z-scanning device specifically includes the following steps:

[0018] S1: Use the translation stage to move the sample to be measured to the focal position z1 of the focusing lens, obtain the wavelength window of the current two-photon fluorescence signal, the fluorescence collection device correspondingly collects the two-photon fluorescence signal from the sample to be measured, and sends the correspondingly collected two-photon fluorescence signal to the fluorescence intensity measurement module; the second energy meter uses the collection lens to correspondingly receive the measurement beam passing through the sample to be measured; the fluorescence intensity measurement module, the first energy meter, and the second energy meter send their respective acquisition results to the energy collection module;

[0019] S2: Control the sample to be measured to move along the optical axis of the focusing lens at a preset step size, repeat step S1 until h sets of measurement data are obtained, and ensure that the wavelength ranges of the two-photon fluorescence signals of the samples to be measured at different positions are all within the wavelength window; the measurement data includes the position z of the sample to be measured, the fluorescence intensity L of the sample to be measured at the current position z, and the pulse energies respectively received by the first energy meter and the second energy meter;

[0020] S3: The computer completes the Z-scanning of the sample to be measured and the measurement of the two-photon fluorescence data based on h sets of measurement data. The two-photon fluorescence data includes the two-photon absorption coefficient and the two-photon absorption cross-section.

[0021] Furthermore, in step S1, the specific steps for obtaining the fluorescence intensity of the sample to be measured at the focal position are as follows:

[0022] S11: Use the translation stage to move the sample to be measured to the focal position z1 of the focusing lens;

[0023] S12: Adjust the position of the fiber collimator to make the fluorescence peak intensity of the two-photon fluorescence signal collected by the fiber collimator the highest, and receive the spectrum Sc of the current two-photon fluorescence signal;

[0024] S13: Obtain the wavelength window of the current two-photon fluorescence signal. If the fluorescence intensity measurement module is a spectrometer, integrate the spectrum within the wavelength window through the following formula to obtain the fluorescence intensity L1 at the focal position, otherwise directly obtain the fluorescence intensity L1 at the focal position using a photodiode:

[0025] (1).

[0026] Furthermore, the specific steps of step S3 include:

[0027] S31: The computer fits the transmittance curve based on h groups of measurement data for fitting;

[0028] S32: Set the objective function as :

[0029] (2);

[0030] (3);

[0031] (4);

[0032] wherein, is the change of the absorption rate of the sample to be measured with the position z, is the change of the fluorescence intensity of the sample to be measured with the position z, is the logarithm of the ratio of the fluorescence intensity to the absorption rate when the sample to be measured is at the position z;

[0033] S33: It can be known from the expression of the transmittance curve that:

[0034] (5);

[0035] (6);

[0036] (7);

[0037] (8);

[0038] (9);

[0039] wherein, is the linear transmittance, λ is the wavelength of the laser pulse, is the beam waist radius, α is the linear absorption coefficient, is the peak pulse optical intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated on the sample to be measured, and are intermediate parameters for calculating the transmittance curve and have no actual physical meaning;

[0040] S34: Use the objective function to complete the optimization of the intermediate parameter ;

[0041] S35: The optimized intermediate parameter Substitute into Equation (8) to obtain the two-photon absorption coefficient β ;

[0042] S36: Calculate the two-photon absorption cross-section of the sample to be measured through the following formula σ (2) :

[0043] (10);

[0044] wherein is the photon energy, N 0 is the particle number density.

[0045] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0046] (1) The two-photon fluorescence Z-scan device and measurement method of the present invention are based on two-photon fluorescence spectroscopy and Z-scan technology to design a measurement method for two-photon absorption cross-section and two-photon fluorescence power density spectrum with low time consumption, low cost and high precision. The components involved in the two-photon fluorescence Z-scan device proposed by the present invention are inexpensive, and have the ability to measure the nonlinear optical properties of the sample to be measured and the two-photon fluorescence power density spectrum under different light intensities within a short time; the present invention solves the problem of high cost of measuring the two-photon absorption cross-section by absolute two-photon fluorescence intensity, realizes the real-time measurement of two-photon fluorescence data varying with light intensity, and can measure the two-photon absorption cross-section through data fitting. In addition, the present invention can improve the sensitivity of measuring the two-photon absorption coefficient of the sample to be measured through the mutual comparison and correction of two different data sources. The purpose of the present invention is to solve the problem of low measurement accuracy of the sensitivity of the two-photon absorption cross-section by combining Z-scan technology and two-photon fluorescence spectroscopy, and reduce the construction cost of the device.

[0047] (2) The two-photon fluorescence Z-scan device and measurement method of the present invention aim to solve the problem of low sensitivity of the existing open-aperture Z-scan technology. The present invention adds a fluorescence collection module to the translation stage of the ordinary open-aperture Z-scan technology, which not only reduces the measurement cost, but also improves the measurement accuracy and sensitivity of the two-photon absorption coefficient / two-photon absorption cross-section. The present invention cleverly designs the objective function, enabling the least squares method to be better applied to the nonlinear fitting algorithm, thereby obtaining better optimization results. The calibration scheme used in the present invention refers to data from the measurement results of the same sample and the same pulse. Therefore, compared with the calibration scheme using literature data in the TPF technology, it has higher practical reference value and is conducive to stronger measurement stability in the measurement of different environments and different batches of samples. Description of the Drawings

[0048] The accompanying drawings, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0049] Figure 1 Schematic diagram of the two-photon fluorescence Z-scan device according to the embodiment of the present invention;

[0050] Figure 2 Flow schematic diagram of the two-photon fluorescence Z-scan measurement method according to the embodiment of the present invention;

[0051] Figure 3 Schematic diagram of the structure of the collection device according to the embodiment of the present invention;

[0052] Figure 4 Schematic diagram of the structure of the integrating sphere collection device according to the embodiment of the present invention;

[0053] Figure 5 Z-scan transmittance curve measured using rhodamine 6G ethanol solution according to the embodiment of the present invention;

[0054] Figure 6 Two-photon fluorescence curve measured using rhodamine 6G ethanol solution according to the embodiment of the present invention;

[0055] Figure 7 Z-scan transmittance curve measured using InP quantum dot toluene solution according to the embodiment of the present invention;

[0056] Figure 8 Two-photon fluorescence curve measured using InP quantum dot toluene solution according to the embodiment of the present invention.

[0057] Explanation of reference numerals:

[0058] 1. Pulse laser; 2. Pulse generator; 3-1. Diaphragm; 3-2. Half-wave plate; 3-3. Beam-splitting prism; 4. Beam-splitting plate; 5. First energy meter; 6. Focusing mirror; 7. Fluorescence collection module; 7-1. Optical fiber; 7-2. Fiber collimator; 7-3. Cuvette slot; 7-4. Integrating sphere; 8. Collection mirror; 9. Second energy meter; 10. Energy collection module; 11. Fluorescence intensity measurement module; 12. Translation stage; 13. Computer; 14. Mirror; 15. Sample to be measured. Detailed implementation manners

[0059] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation to the present invention.

[0060] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0061] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present invention. In addition, the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first", "second", etc. may explicitly or implicitly include one or more of such features. In the description of the present invention, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0062] In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, the terms "install", "connect", "couple" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood through specific situations.

[0063] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0064] Such as Figure 1As shown in the figure, the present invention provides a two-photon fluorescence Z-scan device, which includes a pulsed laser 1, a pulse generator 2, a fluorescence intensity measurement module 11, an attenuation module, a beam splitter 4, a first energy meter 5, a second energy meter 9, an energy collection module 10, a fluorescence collection module 7, a focusing lens 6, a collection lens 8, and a computer 13. Among them, the pulsed laser 1 controls the laser pulse to delay a preset time through the pulse generator 2 to externally trigger the fluorescence intensity measurement module 11, so that the fluorescence intensity measurement module 11 receives the laser pulse and the corresponding two-photon fluorescence signal; the laser pulse emitted by the pulsed laser 1 also enters the beam splitter 4 through the attenuation module for beam splitting. One beam of laser is received by the first energy meter 5 as a reference beam, and the other beam of laser is irradiated onto the sample to be measured 15 through the focusing lens 6 as a measurement beam. The sample to be measured 15 generates a two-photon fluorescence signal under the excitation of the measurement beam. The fluorescence collection device collects the two-photon fluorescence signal and sends the collected two-photon fluorescence signal to the fluorescence intensity measurement module 11. The measurement beam passing through the sample to be measured 15 is received by the second energy meter 9 through the collection lens 8;

[0065] The sample to be measured 15 moves along the optical axis of the focusing lens 6 at a preset step size. The fluorescence collection device is relatively stationary with respect to the sample to be measured 15, and correspondingly collects the two-photon fluorescence signal from the sample to be measured 15, and sends the correspondingly collected two-photon fluorescence signal to the fluorescence intensity measurement module 11. The second energy meter 9 uses the collection lens 8 to correspondingly receive the measurement beam passing through the sample to be measured 15; the first energy meter 5 and the second energy meter 9 send their respective collection results to the energy collection module 10; the computer 13 completes the Z-scan of the sample to be measured 15 and the measurement of the two-photon fluorescence data according to the data obtained by the energy collection module 10 and the fluorescence intensity measurement module 11 respectively.

[0066] It should be noted that a reflector 14 can be set on the optical path of the measurement beam from the beam splitter 4 to the focusing lens 6 as needed to fold the optical path.

[0067] The present invention combines the Z-scan technology with the two-photon fluorescence spectroscopy technology to measure the transmittance, two-photon fluorescence spectrum, and laser scattering spectrum of the sample to be measured 15 at different positions on the optical axis. Among them, due to the nonlinear absorption effect, the intensity of the two-photon fluorescence signal (two-photon excited fluorescence) is different at different positions, so that the fluorescence intensity shows a mountain-shaped curve with the change of the position of the sample to be measured 15, and the two-photon absorption cross section can be calculated according to this curve.

[0068] In some embodiments, the fluorescence intensity measurement module 11 is a combination of a band-pass filter and a photodiode or a spectrometer. If the fluorescence intensity measurement module 11 uses a combination of a band-pass filter and a photodiode, the total number of electrons measured by the photodiode is the fluorescence intensity.

[0069] It should be noted that the acquisition of two-photon fluorescence signals is completed by a spectrometer, which is costly. In the actual production process, a time-delay circuit with a fixed delay can be used, and the spectrometer can be replaced with a combination of a band-pass filter and a photodiode, thereby greatly reducing the cost of the fluorescence intensity measurement module 11. If a combination of a band-pass filter and a photodiode is used, the spectral integration step in the data processing procedure can be omitted, that is, the total number of electrons measured by the photodiode is the fluorescence intensity L.

[0070] In some embodiments, the attenuation module includes a diaphragm 3-1, a half-wave plate 3-2, and a beam splitter prism 3-3 arranged in sequence along the optical axis. The attenuation module is used to attenuate and control the energy of the laser pulse.

[0071] In some embodiments, the fluorescence collection module 7 is a collection device or an integrating sphere collection device. Among them, the collection device is composed of an optical fiber collimator 7-2 and an optical fiber 7-1 connected in sequence. The optical fiber collimator 7-2 collects the two-photon fluorescence signal generated by the excitation of the sample to be measured 15 by the measurement beam, and the optical fiber 7-1 sends the two-photon fluorescence signal to the fluorescence intensity measurement module 11;

[0072] The integrating sphere collection device includes an optical fiber collimator 7-2, an optical fiber 7-1, an integrating sphere 7-4, and a cuvette slot 7-3 built in the integrating sphere 7-4. The optical fiber 7-1 is connected to the optical fiber collimator 7-2. The cuvette slot 7-3 is used to place the sample to be measured 15. The measurement beam irradiates the sample to be measured 15. The optical fiber collimator 7-2 collects the two-photon fluorescence signal generated by the excitation of the sample to be measured 15 by the measurement beam, and the optical fiber 7-1 sends the two-photon fluorescence signal to the fluorescence intensity measurement module 11.

[0073] In some embodiments, the two-photon fluorescence Z-scan device further includes a translation stage 12, and the translation stage 12 is used to adjust the positions of the sample to be measured 15 and the fluorescence collection module 7.

[0074] As Figure 2As shown in the figure, the present invention proposes a two-photon fluorescence Z-scan measurement method, which is realized by a two-photon fluorescence Z-scan device, and specifically includes the following steps: S1: Use the translation stage 12 to move the sample to be measured 15 to the focal position z1 of the focusing mirror 6, obtain the wavelength window of the current two-photon fluorescence signal, the fluorescence collection device correspondingly collects the two-photon fluorescence signal from the sample to be measured 15, and sends the correspondingly collected two-photon fluorescence signal to the fluorescence intensity measurement module 11; the second energy meter 9 uses the collection mirror 8 to correspondingly receive the measurement beam passing through the sample to be measured 15; the fluorescence intensity measurement module 11, the first energy meter 5 and the second energy meter 9 send their respective acquisition results to the energy collection module 10; S2: Control the sample to be measured 15 to move along the optical axis of the focusing mirror 6 at a preset step size, repeat step S1 until h groups of measurement data are obtained, and make the wavelength ranges of the two-photon fluorescence signals of the samples to be measured 15 at different positions all within the wavelength window; the measurement data includes the position z of the sample to be measured 15, the fluorescence intensity L of the sample to be measured 15 at the current position z, and the pulse energies respectively received by the first energy meter 5 and the second energy meter 9; S3: The computer 13 completes the Z-scan of the sample to be measured 15 and the measurement of the two-photon fluorescence data based on h groups of measurement data, and the two-photon fluorescence data includes the two-photon absorption coefficient and the two-photon absorption cross section.

[0075] In some embodiments, in step S1, the specific steps of obtaining the fluorescence intensity of the sample to be measured 15 at the focal position are as follows:

[0076] S11: Use the translation stage 12 to move the sample to be measured 15 to the focal position z1 of the focusing mirror 6;

[0077] S12: Adjust the position of the fiber collimator 7-2 to make the fluorescence peak intensity of the two-photon fluorescence signal collected by the fiber collimator 7-2 the highest, and receive the spectrum Sc of the current two-photon fluorescence signal;

[0078] S13: Obtain the wavelength window of the current two-photon fluorescence signal. If the fluorescence intensity measurement module 11 is a spectrometer, integrate the spectrum within the wavelength window through the following formula to obtain the fluorescence intensity L1 at the focal position, otherwise directly obtain the fluorescence intensity L1 at the focal position using a photodiode:

[0079] (1).

[0080] In some embodiments, the specific steps of step S3 include:

[0081] S31: The computer 13 fits the transmittance curve based on h groups of measurement data;

[0082] S32: Set the objective function as :

[0083] (2);

[0084] (3);

[0085] (4);

[0086] wherein, is the change of the absorption rate of the sample 15 to be measured with respect to the position z, is the change of the fluorescence intensity of the sample 15 to be measured with respect to the position z, is the logarithm of the ratio of the fluorescence intensity to the absorption rate when the sample 15 to be measured is at the position z;

[0087] S33: From the expression of the transmittance curve it can be known that:

[0088] (5);

[0089] (6);

[0090] (7);

[0091] (8);

[0092] (9);

[0093] wherein, is the linear transmittance, λ is the wavelength of the laser pulse, is the beam waist radius, α is the linear absorption coefficient, is the peak pulse optical intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated on the sample 15 to be measured, and are intermediate parameters for calculating the transmittance curve and have no actual physical meaning;

[0094] S34: Use the objective function to complete the optimization of the intermediate parameter ;

[0095] S35: Substitute the optimized intermediate parameter into Equation (8) to obtain the two-photon absorption coefficient β ;

[0096] S36: Calculate the two-photon absorption cross section of the sample 15 to be measured through the following formulaσ (2) :

[0097] (10);

[0098] Wherein, is the photon energy, N 0 is the particle number density.

[0099] In some embodiments, in step S31, the calculation steps of the transmittance T of the sample 15 to be measured at different positions are as follows:

[0100] Without placing the sample 15 to be measured, calculate the pulse energy ratio R received by the first energy meter 5 and the second energy meter 9 E ;

[0101] After placing the sample, calculate the pulse energy ES1 irradiated to the sample 15 to be measured at the current position based on the pulse energy received by the first energy meter 5: ES1 = E11×R E ; where E11 is the pulse energy received by the first energy meter 5;

[0102] Calculate the transmittance T based on the pulse energy received by the second energy meter 9 and the pulse energy ES1 irradiated to the sample 15 to be measured at the current position: T1 = E21 / ES1; where E21 is the pulse energy received by the second energy meter 9.

[0103] It should be noted that the fitting of the transmittance curve of Z-scan is used as a reference (due to the phenomenon that some samples have no fluorescence), which is mutually verified with two-photon fluorescence Z-scan. If signals appear in both, the two-photon absorption coefficient can be calculated independently.

[0104] Embodiment 1

[0105] The embodiment uses a Q-smart450 Nd: YAG pulsed laser 1, the repetition frequency of the pulsed laser 1 is 10Hz, the working center wavelength is 1064nm, the pulse duration is 6ns, the beam splitter prism 3-3 uses a polarization beam splitter prism 3-3, the model of the beam splitter 4 is BP145B3, (beam splitting ratio 45:55, R:T), the models of the first energy meter 5 and the second energy meter 9 are both PE9-C, the pulse controller uses DG645, and the fluorescence intensity measurement module 11 is a spectrometer.

[0106] The pulsed laser 1 controls the pulse delay to be 140 μs outside the DG645 to trigger the spectrometer externally, ensuring that the spectrometer can receive each laser pulse and the corresponding two-photon fluorescence signal. The laser pulse emitted by the laser passes through the aperture 3-1 and the half-wave plate 3-2 (1 / 2 wave plate), and then the energy is adjusted by the polarization beam splitter prism 3-3 with a single wavelength of 1064 nm. Subsequently, it is divided into two beams by the beam splitter 4. One beam of laser enters the first energy meter 5 for calibrating the energy of the incident laser pulse, and the other beam of laser is focused on the sample to be measured after passing through the 300-mm near-infrared focusing mirror 6. The sample to be measured 15 scans along the optical axis on the electric translation stage 12 at a preset step size. At this time, if there is a two-photon fluorescence signal in the sample to be measured 15, it will be collected by the fiber collimator 7-2 with a focal length of 10 mm that is relatively stationary with respect to the position of the sample to be measured 15, and the two-photon fluorescence signal is transmitted to the spectrometer via the optical fiber 7-1. At the same time, the laser passing through the sample is focused on the second energy meter 9 by the 75-mm general focusing mirror 6 (i.e., the collection mirror 8 with a focal length of 75 mm). The laser energy data obtained by the first energy meter 5 and the second energy meter 9 will also be subjected to analog-to-digital conversion by the energy collection module 10 and transmitted to the computer 13 for further data processing, completing the simultaneous measurement of the Z-scan and two-photon fluorescence data.

[0107] After obtaining the energy and two-photon fluorescence spectrum data of the sample to be measured 15 at different positions, the two-photon absorption cross-section of the sample to be measured 15 can be calculated by calibrating with the calibration sample rhodamine 6g reagent. This calibration process is different from the calibration using the absorption coefficient in the TPF method. In the present invention, the same sample and the data of the same measurement are used for calibration, so it is relatively more accurate.

[0108] The way to obtain the Z-scan curve (i.e., the transmittance curve) is as follows: In the case of being empty (without placing the sample to be measured 15), calculate the measurement ratio R of the pulse energy E10 of the first energy meter 5 and the pulse energy E20 of the second energy meter 9 E ; after placing the sample to be measured 15 at a specific position z1, the energy ES1 irradiated on the sample to be measured 15 can be calculated using the pulse energy E11 of the first energy meter 5 and the pulse energy E21 of the second energy meter 9 at this time, where ES1 = E11 × R E , then the transmittance T = E21 / ES1. Each position z of the translation stage 12 corresponds to a transmittance T, and collecting and connecting them is the Z-scan curve.

[0109] In the present invention, a fluorescence collection module 7 for two-photon fluorescence spectrum is installed on the translation stage 12. The fluorescence collection module 7 for two-photon fluorescence spectrum includes a simple collection device and an integrating sphere collection device. As Figures 3 - 4As shown in the figure, the simple collection device consists of an optical fiber collimator 7-2 and an optical fiber 7-1. The collection optical path of the collection device forms an angle with the laser optical path, preferably 90°. When the sample thickness is small, such as in the case of a 1 mm optical path, this angle can be changed to 45° for convenient collection. The simple collection device needs to be operated in a dark external environment. The setup of the integrating sphere collection device is relatively simple, and the collected two-photon fluorescence signal is relatively strong, but the cost is slightly higher. Users can choose one of them independently. The integrating sphere collection device includes a customized integrating sphere 7-4, above which there is a cuvette slot 7-3 that allows the laser beam to just pass through the bottom of the cuvette. The materials of both the cuvette slot 7-3 and the inner wall of the integrating sphere 7-4 are PTFE. The angle between the collection optical path and the laser optical path of the integrating sphere collection device is 90°.

[0110] Due to the diffuse reflection effect inside the integrating sphere 7-4, compared with the simple collection device, the optical fiber collimator 7-2 can collect more two-photon fluorescence signals. Tests have shown that the two-photon fluorescence intensity at the focal point can be increased by 2-3 orders of magnitude. At the same time, because the outer shell of the integrating sphere 7-4 is closed, stray light from the outside is not likely to affect the collection of two-photon fluorescence signals, while when using the simple collection device, the experimental room needs to be kept dark.

[0111] After the translation stage 12 moves to a specific position z1, the first energy meter 5, the second energy meter 9, and the spectrometer will synchronously collect 5 sets of data (each set of data includes an energy value E11 and an energy value E21, and a spectral data S1, which is an array of 2048×1). The data of the first energy meter 5 and the second energy meter 9 are used to calculate the transmittance T, and the spectral data of the spectrometer will go through steps such as background removal, band selection, and fluorescence peak integration to obtain the fluorescence intensity L. After one scan, if the number of moving steps is 200, the scanning position information Z (an array of 200×1), the transmittance T (an array of 200×1), the fluorescence intensity L (an array of 200×1), and the energy ES1 irradiated on the sample (an array of 200×1) can be obtained.

[0112] Using the fluorescence intensity measurement module 11 to collect spectral data will go through steps such as background removal, band selection, and fluorescence peak integration to obtain the fluorescence intensity L. Taking one acquisition at a specific position z1 as an example: first, close the light input port of the spectrometer and measure the background spectrum S0; then open the light input port of the spectrometer and measure the spectrum S1. The background removal operation is to calculate Sc( ) = S1 - S0.

[0113] In the band selection step, the translation stage 12 is moved to the focal position zc, and the position of the fiber collimator 7-2 is adjusted to maximize the fluorescence peak intensity of the received two-photon fluorescence signal. The measured spectrum is denoted as Sc. A wavelength window is selected such that the wavelength range of the two-photon fluorescence signal is [λmin, λmax]. Then, the spectrum within this wavelength range is integrated to obtain the fluorescence intensity Lc at the focal position. Similarly, the expression for the fluorescence intensity L1 collected at a specific position z1 is:

[0114] (1);

[0115] After the computer 13 obtains the position information z, the fluorescence intensity L, and the energy ES1 irradiated on the sample 15 to be measured, it fits the transmittance curve T(z). The transmittance curve T(z) is:

[0116] (5);

[0117] (6);

[0118] (7);

[0119] (8);

[0120] (9);

[0121] Where, T 0 is the linear transmittance, λ is the wavelength of the laser pulse, w 0 is the beam waist radius, which is taken as 116 μm here, α is the linear absorption coefficient, is the peak pulse intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated on the sample 15 to be measured, and are intermediate parameters for calculating the transmittance curve and have no actual physical meaning, β is the two-photon absorption coefficient.

[0122] In actual measurement, generally, the intermediate parameter q 0 is fitted, and then the two-photon absorption coefficient β and the two-photon absorption cross-section σ (2) are calculated from it. Since the reflected and scattered parts can generally be ignored compared to the energy of the laser pulse. Therefore, the energy absorbed by the sample 15 to be measured is proportional to the absorption rate, that is: 。

[0123] Meanwhile, according to the law of conservation of energy, the energy absorbed by the sample to be measured 15 will be converted into second-harmonic photons and released at a certain ratio. Therefore, we have:

[0124] (11);

[0125] Since L ( z ) and A ( z ) differ by a coefficient, the objective function during optimization needs to be modified accordingly.

[0126] Generally, the least squares method is used to set the objective function as:

[0127] (12);

[0128] This is obviously unreasonable because when the magnitudes of L ( z ) and A ( z ) are quite different, this objective function is likely to fall into a local minimum and ignore the data of the weaker two-photon fluorescence signal. We can set:

[0129] (3);

[0130] (2);

[0131] Among them, when L ( z ) and A ( z ) have the same linear shape, that is, they only differ by a coefficient, for all sample positions z, the R L value approaches a constant. Let the sequence R L subtract the largest number max R L , then all the values of the sequence will tend to zero, and finally the least squares method can be used to complete the optimization.

[0132] Similar to the Z-scan curve fitting, generally 0 can be selected as the optimization quantity to calculate the two-photon absorption coefficient. Using a rhodamine 6G ethanol solution as the test sample, the obtained data and fitting results are as shown in q 0. As the test sample, the obtained data and fitting results are as shown in Figures 5 - 6As shown. The fitting results show that the fitting method of the present invention is approximately 2.4 times that of the Z-scan curve fitting. This numerical result indicates that the two-photon absorption coefficient obtained by fitting the two-photon fluorescence curve is indeed larger than the result obtained by fitting the Z-scan curve. This is due to the fact that the signal-to-noise ratio of two-photon fluorescence is higher than that of Z-scan. At the same time, since Z-scan measures the transmittance, for highly transmissive media, in order to obtain a more accurate change in transmittance, the change amplitude of the laser pulse energy must be quite small. Reflection and scattering will also have a very large impact on the measurement sensitivity (generally, reflection and scattering of the laser are rarely considered in the open-aperture Z-scan). In contrast, the collection and measurement of two-photon fluorescence can easily obtain a result with a higher signal-to-noise ratio by improving the sensitivity of the photosensitive element. Therefore, in some samples (such as Figures 7 - 8 the toluene solution of InP quantum dots shown), the appearance of the two-photon fluorescence curve can be visually observed, but it is relatively difficult to observe the existence of the Z-scan curve, even though its uncertainty can reach about ±0.5%.

[0133] Therefore, for some samples with two-photon fluorescence, the two-photon fluorescence Z-scan measurement method (the method of the present invention) is a method with relatively high sensitivity, which can measure the two-photon absorption coefficient when the open-aperture Z-scan technology fails to successfully measure the signal. Similarly, here, the two-photon absorption coefficient of a specified sample can also be obtained by calibrating the two-photon fluorescence Z-scan of the sample and combining the fitting results of the open-aperture Z-scan, that is, by calibration. For example, in Figures 5 - 6 , the q 0 obtained by using the open-aperture Z-scan technology is 0.06994, and the q 0 obtained by using the two-photon fluorescence Z-scan measurement method is 0.1745; while in Figures 7 - 8 , it is difficult to obtain an accurate q 0 value by using the open-aperture Z-scan technology, and the q 0 obtained by the two-photon fluorescence Z-scan measurement method is 0.03433. Then, it can be inferred that for this sample, the q 0 obtained by using the open-aperture Z-scan technology should be 0.01376, corresponding to a 0.68% reduction in transmittance at the focus. Since this change in transmittance is too low compared to the sensitivity of the existing open-aperture Z-scan system, it is very difficult to be observed.

[0134] It should be noted here that the data involved in this calibration process are the results of real-time measurement of the calibration sample. From the measurement results, it can be seen that the two-photon fluorescence Z-scan measurement method has relatively low requirements for the standardization of the calibration sample (including pharmaceutical preparation process, sample conditions, test conditions, etc.) while ensuring accuracy, and only requires the simultaneous presence of obvious two-photon absorption and two-photon fluorescence phenomena, and has a higher cost performance compared with the TPF technology.

[0135] It should be understood that the various forms of processes shown above can be used, with steps reordered, added or deleted. For example, the steps described in the disclosure of the present invention can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and no limitation is made herein.

[0136] The above specific embodiments do not constitute a limitation on the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub - combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A two-photon fluorescence Z-scan measurement method, which is realized by a two-photon fluorescence Z-scan device, characterized in that: Specifically, it includes the following steps: S1: Use a translation stage to move the sample to be measured to the focal position z1 of the focusing lens, obtain the wavelength window of the current two-photon fluorescence signal. The fluorescence collection device correspondingly collects the two-photon fluorescence signal from the sample to be measured, and sends the correspondingly collected two-photon fluorescence signal to the fluorescence intensity measurement module; The second energy meter uses a collection mirror to correspondingly receive the measurement beam passing through the sample to be measured; The fluorescence intensity measurement module, the first energy meter and the second energy meter send their respective acquisition results to the energy collection module; S2: Control the sample to be measured to move along the optical axis of the focusing lens at a preset step size, repeat step S1 until h groups of measurement data are obtained, and make the wavelength ranges of the two-photon fluorescence signals of the samples to be measured at different positions all within the wavelength window; The measurement data includes the position z of the sample to be measured, the fluorescence intensity L of the sample to be measured at the current position z, and the pulse energies respectively received by the first energy meter and the second energy meter; S3: The computer completes the Z-scan of the sample to be measured and the measurement of the two-photon fluorescence data based on h groups of measurement data. The two-photon fluorescence data includes the two-photon absorption coefficient and the two-photon absorption cross-section; S31: The computer fits the transmittance curve based on h groups of measurement data for fitting; S32: Set the objective function to : （1）； （2）； （3）； Among them, is the change of the absorption rate of the sample to be measured with the position z, is the change of the fluorescence intensity of the sample to be measured with the position z, is the logarithm of the ratio of the fluorescence intensity to the absorption rate when the sample to be measured is at the position z, is the logarithm of the ratio of the fluorescence intensity to the absorption rate; S33: From the expression of the transmittance curve it can be seen that: （4）； （5）； （6）； （7）； （8）； Among them, is the linear transmittance, λ is the wavelength of the laser pulse, is the beam waist radius, α is the linear absorption coefficient, is the peak pulse optical intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated on the sample to be measured, and are intermediate parameters for calculating the transmittance curve and have no physical meaning; S34: Optimize the intermediate parameter using the objective function ; S35: Substitute the optimized intermediate parameters into Equation (7) to obtain the two-photon absorption coefficient β ; S36: Calculate the two-photon absorption cross-section of the sample to be measured by the following formula σ (2) : （9）； Among them, is the photon energy, N 0 is the particle number density.

2. The two-photon fluorescence Z-scan method according to claim 1, characterized in that: The two-photon fluorescence Z-scan device includes a pulsed laser, a pulse generator, a fluorescence intensity measurement module, an attenuation module, a beam splitter, a first energy meter, a second energy meter, an energy collection module, a fluorescence collection module, a focusing lens, a collection mirror and a computer. Among them, the pulsed laser controls the laser pulse to delay a preset time through the pulse generator to externally trigger the fluorescence intensity measurement module, so that the fluorescence intensity measurement module receives the laser pulse and the corresponding two-photon fluorescence signal; The laser pulse emitted by the pulsed laser also enters the beam splitter through the attenuation module for beam splitting. One beam of laser is received by the first energy meter as a reference beam, and the other beam of laser is used as a measurement beam and irradiated onto the sample to be measured through the focusing lens. The sample to be measured is excited by the measurement beam to generate a two-photon fluorescence signal. The fluorescence collection device collects the two-photon fluorescence signal and sends the collected two-photon fluorescence signal to the fluorescence intensity measurement module. The measurement beam passing through the sample to be measured is received by the second energy meter through the collection mirror; The sample to be measured moves along the optical axis of the focusing lens at a preset step size. The fluorescence collection device is relatively stationary with respect to the sample to be measured, correspondingly collects the two-photon fluorescence signal from the sample to be measured, and sends the correspondingly collected two-photon fluorescence signal to the fluorescence intensity measurement module. The second energy meter uses the collection mirror to correspondingly receive the measurement beam passing through the sample to be measured; The first energy meter and the second energy meter send their respective acquisition results to the energy collection module; The computer completes the Z-scan of the sample to be measured and the measurement of the two-photon fluorescence data according to the data respectively obtained by the energy collection module and the fluorescence intensity measurement module.

3. The two-photon fluorescence Z-scan method according to claim 2, wherein: The fluorescence intensity measurement module is a combination of a band-pass filter and a photodiode or a spectrometer. If the fluorescence intensity measurement module uses a combination of a band-pass filter and a photodiode, the total number of electrons measured by the photodiode is the fluorescence intensity.

4. The two-photon fluorescence Z-scan method according to claim 2, wherein: The attenuation module includes a diaphragm, a half-wave plate, and a beam splitter prism arranged in sequence along the optical axis. The attenuation module is used to attenuate and control the energy of laser pulses.

5. The two-photon fluorescence Z-scan method according to claim 2, wherein: The fluorescence collection module is a collection device or an integrating sphere collection device. Among them, the collection device is composed of an optical fiber collimator and an optical fiber connected in sequence. The optical fiber collimator collects the two-photon fluorescence signal generated by the measurement beam exciting the sample to be measured, and the optical fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module; The integrating sphere collection device includes an optical fiber collimator, an optical fiber, an integrating sphere, and a cuvette slot built in the integrating sphere. The optical fiber is connected to the optical fiber collimator. The cuvette slot is used to place the sample to be measured. The measurement beam irradiates the sample to be measured. The optical fiber collimator collects the two-photon fluorescence signal generated by the measurement beam exciting the sample to be measured, and the optical fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module.

6. The two-photon fluorescence Z-scan method according to claim 3, wherein: The two-photon fluorescence Z-scan device further includes a translation stage, and the translation stage is used to adjust the positions of the sample to be measured and the fluorescence collection module.

7. The two-photon fluorescence Z-scan method according to claim 1, characterized in that: In step S1, the specific steps for obtaining the fluorescence intensity of the sample to be measured at the focal position are as follows: S11: Use the translation stage to move the sample to be measured to the focal position z1 of the focusing lens; S12: Adjust the position of the optical fiber collimator to make the fluorescence peak intensity of the two-photon fluorescence signal collected by the optical fiber collimator the highest, and receive the spectrum Sc of the current two-photon fluorescence signal; S13: Obtain the wavelength window of the current two-photon fluorescence signal. If the fluorescence intensity measurement module is a spectrometer, integrate the spectrum within the wavelength window through the following formula to obtain the fluorescence intensity L1 at the focal position. Otherwise, directly obtain the fluorescence intensity L1 at the focal position using a photodiode: （10）。

Images