Two-photon fluorescence Z scanning device and measuring method

Abstract

The invention belongs to the technical field of nonlinear optics, and particularly relates to a two-photon fluorescence Z scanning device and a measurement method. The method comprises the following steps: S1, moving a to-be-detected sample to a focus position z1 of a focus lens by using a translation stage, obtaining a wavelength window of a current two-photon fluorescence signal, and correspondingly collecting the two-photon fluorescence signal from the to-be-detected sample by using a fluorescence collection device; the fluorescence intensity measurement module, the first energy meter and the second energy meter send respective collection results to the energy collection module; s2, controlling the to-be-measured sample to move along the optical axis of the focus lens according to a preset step length, and repeating the step S1 until h groups of measurement data are obtained; s3, the computer completes Z scanning of the to-be-measured sample and measurement of two-photon fluorescence data based on the h groups of measurement data. The device has the capability of measuring the two-photon fluorescence intensity and the two-photon absorption coefficient under different light intensities.

Description

Technical Field

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

[0002] The two-photon fluorescence (TPF) process has a wide range of applications in many fields, including but not limited to bioimaging, 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, and can provide high-resolution three-dimensional images; two-photon fluorescence probe technology can be used to study new materials, especially the optical properties of nanomaterials and polymers; it is also an intuitive detection indicator for studying phenomena such as molecular dynamics and energy transfer. Since the light intensity of two-photon fluorescence is proportional to the light energy absorbed by two-photons, both have the potential for nonlinear coefficient measurement. Among them, as early as 2001, D.A. Oulianov 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 area of ​​the sample and obtain the ADC count intensity, which is proportional to the square of the incident light intensity, and its slope k is proportional to the two-photon absorption coefficient. Therefore, after calibrating 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 the measurement requires the use of a calibration solution, which has strict requirements on the preparation process, optical path setting and laser parameters, and the reference sample in reality is affected by a variety of environmental factors, 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 accuracy of the final measurement.

[0004] The Chinese invention patent entitled "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 testing of in-situ femtosecond Z-scan test, second harmonic test, and fluorescence emission excitation spectrum. The Chinese invention patent entitled "A method for measuring 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 test, but in essence, it 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-section of three dicyanopyridine DCM dyes under the excitation of titanium sapphire laser femtosecond pulses based on the TPF method and the open aperture Z-scan method. The two-photon absorption cross-section of the sample was obtained by approximating the experimental transmission curve with the corresponding model curve. However, the TPA cross-section data measured for the same substance by 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 fluorescence method was 134 GM, which is an order of magnitude higher than the 12.8 GM measured by Z-scan method.

[0006] Similarly, Yaochuan Wang et al. used femtosecond open-aperture Z-scanning and two-photon excitation fluorescence to study the two-photon absorption cross-sections of several three-branched derivatives. The two-photon absorption cross-section values ​​measured by the two methods were compared. The results showed that there were significant 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 excitation fluorescence method was 2.1 to 2.3 times larger than that measured by the open-aperture z-scan method. This difference is likely caused by the systematic errors of the different measurements.

[0007] The above description shows that the two-photon fluorescence method of measuring the absorption cross section has certain advantages in terms of measurement sensitivity compared to the conventional Z-scan method, but there are certain differences in the measurement results of the two methods. Therefore, for materials with two-photon fluorescence, how to use 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 scanning device and a measurement method to solve the problem of high cost of measuring the two-photon absorption cross section by two-photon fluorescence intensity in the prior art. The present invention has the advantages of easy construction 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, but also can improve the measurement sensitivity of the two-photon absorption coefficient by comparing the two sets of data.

[0009] To achieve the above object, the technical solution created by the present invention is implemented as follows: A two-photon fluorescence Z scanning device comprises a pulse 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 mirror, a collecting mirror and a computer, wherein the pulse laser controls the laser pulse delay by 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 pulse laser is also incident on the beam splitter through the attenuation module for light splitting, one laser beam is received by the first energy meter as a reference beam, and the other laser beam is irradiated onto a sample to be tested through the focusing mirror as a measurement beam, the sample to be tested is excited by the measurement beam to generate a two-photon fluorescence signal, a fluorescence collecting device collects the two-photon fluorescence signal, and sends the collected two-photon fluorescence signal to the fluorescence intensity measurement module, and the measurement beam passing through the sample to be tested is received by the second energy meter through the collecting mirror; The sample to be tested moves along the optical axis of the focusing mirror according to a preset step length, the fluorescence collection device and the sample to be tested are relatively stationary, and the two-photon fluorescence signal from the sample to be tested is correspondingly collected, and the corresponding collected two-photon fluorescence signal is sent to the fluorescence intensity measurement module, and the second energy meter uses the collection mirror to receive the measurement light beam passing through the sample to be tested; 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 and two-photon fluorescence data measurement of the sample to be tested according to the data respectively obtained by the energy collection module and the fluorescence intensity measurement module.

[0010] Furthermore, the fluorescence intensity measurement module is a combination of a bandpass filter and a photodiode or a spectrometer. If the fluorescence intensity measurement module adopts a combination of a bandpass filter and a photodiode, the total number of electrons measured by the photodiode is the fluorescence intensity.

[0011] Furthermore, the attenuation module includes an aperture, a half-wave plate and a beam splitter prism which are sequentially arranged along the optical axis, and the attenuation module is used to attenuate and control the energy of the laser pulse.

[0012] Furthermore, the fluorescence collection module is a collection device or an integrating sphere collection device, wherein the collection device is composed of a fiber collimator and an optical fiber connected in sequence, the fiber collimator collects a two-photon fluorescence signal generated by the sample to be tested when excited by the measuring light beam, and the optical fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module; The integrating sphere collection device includes a fiber optic collimator, an optical fiber, an integrating sphere, and a cuvette slot built in the integrating sphere. The optical fiber is connected to the fiber optic collimator. The cuvette slot is used to place the sample to be tested. The measuring light beam is irradiated to the sample to be tested. The fiber optic collimator collects the two-photon fluorescence signal generated by the sample to be tested when excited by the measuring light beam. The optical fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module.

[0013] Furthermore, the two-photon fluorescence Z scanning device also includes a translation stage, which is used to adjust the positions of the sample to be tested and the fluorescence collection module.

[0014] A two-photon fluorescence Z scanning measurement method is implemented using a two-photon fluorescence Z scanning device, and specifically comprises the following steps: S1: Use the translation stage to move the sample to be tested to the focal position z1 of the focusing mirror, 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 tested, and sends the corresponding collected two-photon fluorescence signal to the fluorescence intensity measurement module; the second energy meter uses the collection mirror to receive the measurement light beam passing through the sample to be tested; 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: Control the sample to be tested to move along the optical axis of the focusing mirror according to a preset step length, and repeat step S1 until h groups of measurement data are obtained, and the wavelength range of the two-photon fluorescence signal of the sample to be tested at different positions is within the wavelength window; the measurement data includes the position z of the sample to be tested, the fluorescence intensity L of the sample to be tested at the current position z, and the pulse energy received by the first energy meter and the second energy meter respectively; S3: The computer completes the measurement of the Z scan and two-photon fluorescence data of the sample to be measured based on the h groups of measurement data, and the two-photon fluorescence data includes the two-photon absorption coefficient and the two-photon absorption cross section.

[0015] Furthermore, in step S1, the specific steps of obtaining the fluorescence intensity of the sample to be tested at the focal position are: S11: Use the translation stage to move the sample to be tested to the focal position z1 of the focusing mirror; S12: adjusting the position of the fiber optic collimator so that the fluorescence peak intensity of the two-photon fluorescence signal collected by the fiber optic collimator is the highest, and receiving 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 in the wavelength window by the following formula to obtain the fluorescence intensity L1 at the focal position. Otherwise, use a photodiode to directly obtain the fluorescence intensity L1 at the focal position: (1).

[0016] Furthermore, the specific steps of step S3 include: S31: The computer calculates the transmittance curve based on the h group of measurement data Perform fitting; S32: Set the objective function to : (2); (3); (4); in, is the variation of the absorbance of the sample to be tested with position z, is the change of the fluorescence intensity of the sample to be tested with position z, is the logarithm of the ratio of the fluorescence intensity to the absorbance when the sample to be tested is at position z; S33: From the transmittance curve The expression of : (5); (6); (7); (8); (9); in, T 0 is the linear transmittance, λ is the wavelength of the laser pulse, w 0 is the beam waist radius, α is the linear absorption coefficient, is the pulse peak intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated to the sample to be tested, , and It is an intermediate parameter for calculating the transmittance curve and has no actual physical meaning; S34: Use the objective function to complete the intermediate parameters Optimization; S33: The optimized intermediate parameters Substituting into equation (8), we obtain the two-photon absorption coefficient β ; S35: Calculate the two-photon absorption cross section of the sample to be tested by the following formula σ (2) : (10); in, is the photon energy, N 0 is the particle number density.

[0017] Compared with the prior art, the invention can achieve the following beneficial effects: (1) The present invention creates the two-photon fluorescence Z scanning device and measurement method, and designs a low-time, low-cost and high-precision two-photon absorption cross section and two-photon fluorescence power density spectrum measurement method based on two-photon fluorescence spectroscopy technology and Z scanning technology. The components involved in the two-photon fluorescence Z scanning device proposed by the present invention are low-cost and have the ability to measure the nonlinear optical properties of the sample to be tested and the two-photon fluorescence power density spectrum under different light intensities in 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 real-time measurement of two-photon fluorescence data that changes with light intensity, and can realize the measurement of the two-photon absorption cross section by data fitting. In addition, the present invention can improve the sensitivity of measuring the two-photon absorption coefficient of the sample to be tested by 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 two-photon absorption cross section sensitivity by combining Z scanning technology and two-photon fluorescence spectroscopy technology, and reduce the construction cost of the device.

[0018] (2) The present invention creates the two-photon fluorescence Z scanning device and measurement method to solve the problem of low sensitivity of the existing open-aperture Z scanning technology. The present invention 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 by adding a fluorescence collection module to the translation stage of the ordinary open-aperture Z scanning technology. The present invention cleverly designs the objective function so that the least squares method can be better applied to the nonlinear fitting algorithm, thereby obtaining better optimization results. The calibration scheme reference data used in the present invention is derived from the measurement results of the same sample and the same pulse. Therefore, compared with the TPF technology calibrated using literature data, it has a higher actual reference value, which is beneficial for the technology to show stronger measurement stability in the measurement of different environments and different batches of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation on the present invention. In the drawings: Figure 1 A schematic diagram of a two-photon fluorescence Z scanning device according to an embodiment of the present invention; Figure 2 A schematic diagram of the process of the two-photon fluorescence Z-scanning measurement method according to an embodiment of the present invention; Figure 3 A schematic diagram of the structure of the collecting device according to an embodiment of the present invention; Figure 4 A schematic diagram of the structure of an integrating sphere collection device according to an embodiment of the present invention; Figure 5This is a Z scan transmittance curve obtained by measuring a 6g ethanol solution of rhodamine as described in an embodiment of the present invention; Figure 6 A two-photon fluorescence curve obtained by measuring a 6g ethanol solution of rhodamine as described in an embodiment of the present invention; Figure 7 A Z scan transmittance curve obtained by measuring the InP quantum dot toluene solution according to an embodiment of the present invention; Figure 8 This is a two-photon fluorescence curve obtained by measuring InP quantum dot toluene solution as described in the embodiment of the present invention.

[0020] Description of reference numerals: 1. Pulse laser; 2. Pulse generator; 3-1. Aperture; 3-2. Half-wave plate; 3-3. Beam splitter; 4. Beam splitter; 5. First energy meter; 6. Focusing mirror; 7. Fluorescence collection module; 7-1. Optical fiber; 7-2. Optical fiber collimator; 7-3. Cuvette slot; 7-4. Integrating sphere; 8. Collecting mirror; 9. Second energy meter; 10. Energy collection module; 11. Fluorescence intensity measurement module; 12. Translation stage; 13. Computer; 14. Reflector; 15. Sample to be tested. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the invention more clear, the invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described here are only used to explain the invention and do not constitute a limitation of the invention.

[0022] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0023] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are 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 therefore cannot be understood as a limitation on 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 indicating the number of technical features indicated. Thus, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0024] In the description of the invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installation", "connection" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the invention can be understood according to specific circumstances.

[0025] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0026] like Figure 1 As shown, the present invention proposes a two-photon fluorescence Z scanning device, comprising a pulse 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 mirror 6, a collecting mirror 8 and a computer 13, wherein the pulse laser 1 controls the laser pulse delay by 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 pulse laser 1 is also incident on the beam splitter 4 through the attenuation module for splitting, one laser beam is received by the first energy meter 5 as a reference beam, and the other laser beam is irradiated onto the sample to be tested 15 through the focusing mirror 6 as a measurement beam, and the sample to be tested 15 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 11, and the measurement beam passing through the sample to be tested 15 is received by the second energy meter 9 through the collecting mirror 8; The sample to be tested 15 moves along the optical axis of the focusing mirror 6 according to a preset step size, the fluorescence collection device and the sample to be tested 15 are relatively stationary, and the two-photon fluorescence signal from the sample to be tested 15 is correspondingly collected, and the corresponding collected two-photon fluorescence signal is sent to the fluorescence intensity measurement module 11, and the second energy meter 9 uses the collecting mirror 8 to receive the measurement light beam passing through the sample to be tested 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 and two-photon fluorescence data measurement of the sample to be tested 15 according to the data respectively obtained by the energy collection module 10 and the fluorescence intensity measurement module 11.

[0027] It should be noted that a reflector 14 may be provided on the optical path of the measuring light beam incident from the beam splitter 4 to the focusing mirror 6 as required to fold the optical path.

[0028] The present invention combines Z scanning technology with two-photon fluorescence spectroscopy technology to measure the transmittance, two-photon fluorescence spectrum and laser scattering spectrum of the sample 15 to be tested at different positions on the optical axis. Due to the nonlinear absorption effect, the intensity of the two-photon fluorescence signal (two-photon excited fluorescence) at different positions is different, so that the fluorescence intensity changes with the position of the sample 15 to be tested and presents a peak-shaped curve, and the two-photon absorption cross section can be calculated based on the curve.

[0029] In some embodiments, the fluorescence intensity measurement module 11 is a combination of a bandpass filter and a photodiode or a spectrometer. If the fluorescence intensity measurement module 11 uses a combination of a bandpass filter and a photodiode, the total number of electrons measured by the photodiode is the fluorescence intensity.

[0030] It should be noted that the acquisition of two-photon fluorescence signals is completed by a spectrometer, which is expensive. In the actual production process, a fixed delay circuit can be used, and the spectrometer can be replaced with a combination of a bandpass filter and a photodiode, thereby greatly reducing the cost of the fluorescence intensity measurement module 11. If a combination of a bandpass filter and a photodiode is used, the spectrum integration step in the data processing step can be ignored, that is, the total number of electrons measured by the photodiode is the fluorescence intensity L.

[0031] In some embodiments, the attenuation module includes an aperture 3-1, a half-wave plate 3-2, and a beam splitter prism 3-3 sequentially arranged along the optical axis, and the attenuation module is used to attenuate and control the energy of the laser pulse.

[0032] In some embodiments, the fluorescence collection module 7 is a collection device or an integrating sphere collection device, wherein the collection device is composed of a fiber collimator 7-2 and an optical fiber 7-1 connected in sequence, the fiber collimator 7-2 collects the two-photon fluorescence signal generated by the sample 15 to be tested when excited by the measuring light beam, and the optical fiber 7-1 sends the two-photon fluorescence signal to the fluorescence intensity measurement module 11; The integrating sphere collection device includes a fiber optic 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 fiber optic collimator 7-2. The cuvette slot 7-3 is used to place the sample 15 to be tested. The measuring light beam is irradiated to the sample 15 to be tested. The fiber optic collimator 7-2 collects the two-photon fluorescence signal generated by the sample 15 to be tested when excited by the measuring light beam. The optical fiber 7-1 sends the two-photon fluorescence signal to the fluorescence intensity measurement module 11.

[0033] In some embodiments, the two-photon fluorescence Z scanning device further includes a translation stage 12 , and the translation stage 12 is used to adjust the positions of the sample to be tested 15 and the fluorescence collection module 7 .

[0034] like Figure 2As shown, the present invention proposes a two-photon fluorescence Z scanning measurement method, which is implemented by using a two-photon fluorescence Z scanning device, and specifically includes the following steps: S1: using a translation stage 12 to move the sample 15 to the focal position z1 of the focusing mirror 6, and obtaining the wavelength window of the current two-photon fluorescence signal, the fluorescence collection device correspondingly collects the two-photon fluorescence signal from the sample 15 to be tested, and sends the corresponding collected two-photon fluorescence signal to the fluorescence intensity measurement module 11; the second energy meter 9 uses the collecting mirror 8 to receive the measurement light beam passing through the sample 15 to be tested; the fluorescence intensity measurement module 11, the first energy meter 5 and the second energy meter 9 send their respective collection results to the energy meter 11; The measurement module 10 is a flow chart of a quantity collection module 10; S2: controlling the sample 15 to be tested to move along the optical axis of the focusing mirror 6 at a preset step length, repeating step S1, until h groups of measurement data are obtained, and the wavelength range of the two-photon fluorescence signal of the sample 15 to be tested at different positions is within the wavelength window; the measurement data include the position z of the sample 15 to be tested, the fluorescence intensity L of the sample 15 to be tested at the current position z, and the pulse energy received by the first energy meter 5 and the second energy meter 9 respectively; S3: the computer 13 completes the Z scan and two-photon fluorescence data measurement of the sample 15 to be tested based on the h groups of measurement data, and the two-photon fluorescence data include the two-photon absorption coefficient and the two-photon absorption cross section.

[0035] In some embodiments, in step S1, the specific steps of obtaining the fluorescence intensity of the sample 15 to be tested at the focal position are: S11: Use the translation stage 12 to move the sample 15 to the focal position z1 of the focusing mirror 6; S12: adjusting the position of the optical fiber collimator 7-2 so that the fluorescence peak intensity of the two-photon fluorescence signal collected by the optical fiber collimator 7-2 is the highest, and receiving 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 11 is a spectrometer, integrate the spectrum in the wavelength window by the following formula to obtain the fluorescence intensity L1 at the focal position. Otherwise, directly obtain the fluorescence intensity L1 at the focal position by using a photodiode: (1).

[0036] In some embodiments, the specific steps of step S3 include: S31: The computer 13 calculates the transmittance curve based on the h groups of measurement data. Perform fitting; S32: Set the objective function to : (2); (3); (4); in, is the variation of the absorbance of the sample 15 to be tested with position z, is the change of the fluorescence intensity of the sample 15 to be tested with position z, is the logarithm of the ratio of the fluorescence intensity to the absorbance when the sample 15 to be tested is located at position z; S33: From the transmittance curve The expression of : (5); (6); (7); (8); (9); in, T 0 is the linear transmittance, λ is the wavelength of the laser pulse, w 0 is the beam waist radius, α is the linear absorption coefficient, is the pulse peak intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated to the sample 15 to be tested, , and It is an intermediate parameter for calculating the transmittance curve and has no actual physical meaning; S34: Use the objective function to complete the intermediate parameters Optimization; S33: The optimized intermediate parameters Substituting into equation (8), we obtain the two-photon absorption coefficient β ; S35: Calculate the two-photon absorption cross section of the sample 15 to be tested by the following formula σ (2) : (10); in, is the photon energy, N 0 is the particle number density.

[0037] In some embodiments, in step S31, the calculation steps of the transmittance T of the sample 15 to be tested at different positions are: Without placing the sample 15 to be tested, calculate the pulse energy ratio R received by the first energy meter 5 and the second energy meter 9. E ; After placing the sample, the pulse energy ES1 of the sample 15 to be tested irradiated to the current position is calculated based on the pulse energy received by the first energy meter 5: ES1=E11×R E ; Wherein, E11 is the pulse energy received by the first energy meter 5; The transmittance T is calculated based on the pulse energy received by the second energy meter 9 and the pulse energy ES1 of the sample to be tested 15 irradiated to the current position: T1=E21 / ES1; wherein E21 is the pulse energy received by the second energy meter 9.

[0038] It should be noted that the Z scan transmittance curve fitting is used as a reference (because some samples have no fluorescence) and is verified by the two-photon fluorescence Z scan. If both have signals, the two-photon absorption coefficient can be calculated independently.

[0039] Example 1 The embodiment adopts Q-smart450 Nd: YAG pulse laser 1, the repetition frequency of the pulse laser 1 is 10Hz, the working center wavelength is 1064nm, the pulse duration is 6ns, the beam splitter prism 3-3 adopts a polarization beam splitter prism 3-3, the beam splitter 4 is of model BP145B3 (splitting ratio 45:55, R:T), the first energy meter 5 and the second energy meter 9 are both of model PE9-C, the pulse controller adopts DG645, and the fluorescence intensity measurement module 11 is a spectrometer.

[0040] The pulse laser 1 triggers the spectrometer beyond 140μs by controlling the pulse delay of DG645 to ensure 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 passes through the 1064nm single-wavelength polarization beam splitter prism 3-3 to adjust the energy, and then is divided into two beams through the beam splitter 4. One laser beam enters the first energy meter 5 for the calibration of the incident laser pulse energy, and the other laser beam is focused by the 300mm near-infrared focusing mirror 6 and hits the sample to be tested. The sample to be tested 15 is scanned along the optical axis according to the preset step length on the electric translation stage 12. At this time, if the sample to be tested 15 has a two-photon fluorescence signal, it will be detected by the sample to be tested. The sample 15 is relatively stationary and is collected by the fiber optic collimator 7-2 with a focal length of 10mm. 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 via a 75mm universal focusing mirror 6 (i.e., the collecting mirror 8 with a focal length of 75mm). The laser energy data obtained by the first energy meter 5 and the second energy meter 9 will also be converted from digital to analog by the energy collection module 10 and transmitted to the computer 13 for further data processing, thereby completing the simultaneous measurement of Z scanning and two-photon fluorescence data.

[0041] After obtaining the energy and two-photon fluorescence spectrum data of the sample 15 to be tested at different positions, the two-photon absorption cross section of the sample 15 to be tested can be calculated based on the calibration sample rhodamine 6g reagent. This calibration process is different from the calibration using the absorption coefficient in the TPF method. The present invention uses the same sample and the same measurement data for calibration, so it is relatively more accurate.

[0042] The Z scanning curve (i.e., transmittance curve) is obtained by calculating the ratio R between the pulse energy E10 of the first energy meter 5 and the pulse energy E20 of the second energy meter 9 when the device is empty (without the sample 15 to be tested). E After placing the sample 15 at a specific position z1, the energy ES1 = E11 × R irradiated to the sample 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. 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 scanning curve.

[0043] The present invention installs a fluorescence collection module 7 of two-photon fluorescence spectrum on the translation stage 12, and the fluorescence collection module 7 of two-photon fluorescence spectrum includes a simple collection device and an integrating sphere collection device. Figure 3-Figure 4 As shown, the simple collection device is composed of a fiber collimator 7-2 and an optical fiber 7-1, and the collection light path of the collection device forms an angle with the laser light path, which is recommended to be 90°. When the sample thickness is small, such as 1mm optical path, the angle can be changed to 45° to facilitate collection. The simple collection device needs to be carried out in an environment where the outside is kept in a dark environment. The setting of the integrating sphere collection device is relatively simple, and the collected two-photon fluorescence signal is stronger, but the cost is slightly more expensive, and the user can choose one of them independently. The integrating sphere collection device includes a customized integrating sphere 7-4, on which there is a cuvette slot 7-3, which can make the laser beam just pass through the bottom of the cuvette. The materials of the cuvette slot 7-3 and the inner liner of the integrating sphere 7-4 are both PTFE, and the collection light path of the integrating sphere collection device and the laser light path are at an angle of 90°.

[0044] Due to the diffuse reflection effect inside the integrating sphere 7-4, the fiber optic collimator 7-2 can collect more two-photon fluorescence signals compared to a simple collection device. Tests have shown that the two-photon fluorescence intensity at the focus can be increased by 2-3 orders of magnitude. At the same time, since the outer shell of the integrating sphere 7-4 is closed, external stray light is not easy to affect the collection of two-photon fluorescence signals, while the experimental room must be kept dark when using a simple collection device.

[0045] After the translation stage 12 moves to the 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 energy value E11 and energy value E21, and a spectrum data S1, which is a 2048×1 array), where the data of the first energy meter 5 and the second energy meter 9 are used to calculate the transmittance T, and the spectrum data of the spectrometer will be obtained through background removal, band selection, fluorescence peak integration and other steps to obtain the fluorescence intensity L. After scanning once, if the number of moving steps is 200, the scanning position information Z (200×1 array), transmittance T (200×1 array), fluorescence intensity L (200×1 array), and energy ES1 irradiated to the sample (200×1 array) can be obtained.

[0046] The fluorescence intensity measurement module 11 is used to collect spectrum data, and the fluorescence intensity L is obtained through background removal, band selection, fluorescence peak integration and other steps. Take a collection at a specific position z1 as an example: first close the light inlet of the spectrometer and measure the background spectrum S0; then open the light inlet of the spectrometer and measure the spectrum S1. The background removal operation is to calculate Sc( )=S1-S0.

[0047] 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 make the fluorescence peak intensity of the received two-photon fluorescence signal the highest. The measured spectrum is recorded as Sc, and the wavelength window is selected so that the wavelength range of the two-photon fluorescence signal is [λmin, λmax]. Then the spectrum within the wavelength range is integrated to obtain the fluorescence intensity Lc at the focal position. Similarly, the fluorescence intensity L1 collected at a specific position z1 is expressed as: (1); After the computer 13 obtains the position information z of the sample 15 to be tested, the fluorescence intensity L, and the energy ES1 irradiated to the sample, the transmittance curve T(z) is fitted. The transmittance curve T(z) is: (5); (6); (7); (8); (9); in, T 0 is the linear transmittance, λ is the wavelength of the laser pulse, w 0 is the beam waist radius, which is 116μm here. α is the linear absorption coefficient, is the pulse peak intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated to the sample 15 to be tested, , and It is an intermediate parameter for calculating the transmittance curve and has no actual physical meaning. β is the two-photon absorption coefficient.

[0048] In actual measurement, the intermediate parameters are usually q 0 is fitted, and then the two-photon absorption coefficient is calculated β and two-photon absorption cross section σ (2) Compared with the energy of the laser pulse, the reflected and scattered parts can generally be ignored. Therefore, the energy absorbed by the sample 15 to be tested is proportional to the absorption rate, that is: .

[0049] At the same time, according to the law of conservation of energy, the energy absorbed by the sample 15 to be tested will be converted into frequency-doubled photons and released at a certain ratio, so: (11); because L ( z )and A ( z ) differs by a coefficient, so it is necessary to modify the objective function during optimization.

[0050] Generally, the least squares method is used to solve the objective function Set to: (12); This is obviously unreasonable because L ( z )and A ( z ) differ greatly in magnitude, the objective function can easily fall into a local minimum and ignore the data of weaker two-photon fluorescence signals. You can set: (3); (2); Among them, when L ( z )and A ( z ) have the same linear shape, that is, only one coefficient is different, for all sample positions z R L The value of approaches a constant. Let the sequence R L Subtract the largest number max R L, all the values ​​of the sequence will tend to zero, and finally the least squares method can be used to complete the optimization.

[0051] Same as Z scan curve fitting, generally you can choose q 0 is used as the optimization value to calculate the two-photon absorption coefficient. Using 6g of rhodamine ethanol solution as the test sample, the obtained data and fitting results are shown in Figure 5-Figure 6 As shown. The fitting results show that the fitting method of the present invention is about 2.4 times that of the Z-scan curve fitting. This numerical result illustrates that the two-photon absorption coefficient obtained by two-photon fluorescence curve fitting is indeed larger than the result obtained by Z-scan curve fitting. This is because the signal-to-noise ratio of two-photon fluorescence is higher than that of Z-scan. At the same time, since the Z-scan measures the transmittance, and for highly transparent media, in order to obtain more accurate transmittance changes, the energy variation amplitude of the laser pulse must be quite small, and reflection and scattering will also have a very large impact on the sensitivity of the measurement (generally, open-hole Z-scans rarely consider the reflection and scattering of the laser). In comparison, the collection and measurement of two-photon fluorescence can easily obtain results with a higher signal-to-noise ratio by improving the sensitivity of the photosensitive element. Therefore, in some samples (such as Figure 7-Figure 8 InP quantum dot toluene solution shown in the figure), the appearance of the two-photon fluorescence curve can be directly observed, but the existence of the Z scan curve is difficult to observe, even though its uncertainty can reach about ±0.5%.

[0052] For this reason, for some samples with two-photon fluorescence, the two-photon fluorescence Z-scan measurement method (the method of the present invention) is a relatively sensitive method that can measure the two-photon absorption coefficient when the open-aperture Z-scan technique cannot successfully measure the signal. Similarly, here, the two-photon fluorescence Z-scan of the calibrated sample can be used to obtain the two-photon absorption coefficient of the specified sample in combination with the fitting result of the open-aperture Z-scan, that is, calibration can be performed. For example, Figure 5-Figure 6 The open-aperture Z-scan technique was used to obtain q 0 is 0.06994, obtained using two-photon fluorescence Z-scan measurement method q 0 is 0.1745; Figure 7-Figure 8 It is difficult to obtain accurate q 0 value, while the two-photon fluorescence Z-scan measurement method obtains q 0 is 0.03433, it can be inferred that for this sample, the q 0 should be 0.01376, corresponding to a 0.68% decrease in transmittance at the focus. This transmittance change is too low relative to the sensitivity of the existing open-aperture Z scanning system and is therefore difficult to observe.

[0053] It should be noted here that the data involved in this calibration process are the results of real-time measurement of the calibration samples. From the measurement results, it can be seen that the two-photon fluorescence Z-scan measurement method has relatively low standardization requirements for the calibration samples (including pharmaceutical preparation process, sample conditions, test conditions, etc.) while ensuring accuracy. It only requires obvious two-photon absorption and two-photon fluorescence phenomena at the same time, which is more cost-effective than TPF technology.

[0054] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed 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 this document does not limit this.

[0055] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A two-photon fluorescence Z scanning device, characterized in that: The invention comprises a pulse 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 mirror, a collecting mirror and a computer, wherein the pulse laser controls the laser pulse delay by 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 pulse laser is also incident on the beam splitter through the attenuation module for light splitting, one laser beam is received by the first energy meter as a reference beam, and the other laser beam is irradiated onto the sample to be tested through the focusing mirror as a measurement beam, the sample to be tested 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, and the measurement beam passing through the sample to be tested is received by the second energy meter through the collecting mirror; The sample to be tested moves along the optical axis of the focusing mirror according to a preset step length, the fluorescence collection device is relatively stationary with the sample to be tested, and correspondingly collects two-photon fluorescence signals from the sample to be tested, and sends the corresponding collected two-photon fluorescence signals to the fluorescence intensity measurement module, the second energy meter uses the collection mirror to correspondingly receive the measurement light beam passing through the sample to be tested; 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 and two-photon fluorescence data measurement of the sample to be tested according to the data respectively obtained by the energy collection module and the fluorescence intensity measurement module.

2. The two-photon fluorescence Z scanning device according to claim 1, characterized in that: The fluorescence intensity measurement module is a combination of a bandpass filter and a photodiode or a spectrometer. If the fluorescence intensity measurement module adopts a combination of a bandpass filter and a photodiode, the total number of electrons measured by the photodiode is the fluorescence intensity.

3. The two-photon fluorescence Z scanning device according to claim 1, characterized in that: The attenuation module comprises an aperture, a half-wave plate and a beam splitter prism which are sequentially arranged along the optical axis, and the attenuation module is used for attenuating and controlling the energy of the laser pulse.

4. The two-photon fluorescence Z scanning device according to claim 1, characterized in that: The fluorescence collection module is a collection device or an integrating sphere collection device, wherein the collection device is composed of a fiber collimator and an optical fiber connected in sequence, the fiber collimator collects the two-photon fluorescence signal generated by the sample to be tested under the excitation of the measurement light beam, and the optical fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module; The integrating sphere collection device includes a fiber optic collimator, an optical fiber, an integrating sphere, and a cuvette slot built in the integrating sphere. The optical fiber is connected to the fiber optic collimator. The cuvette slot is used to place the sample to be tested. The measuring light beam is irradiated to the sample to be tested. The fiber optic collimator collects the two-photon fluorescence signal generated by the sample to be tested when excited by the measuring light beam. The optical fiber sends the two-photon fluorescence signal to the fluorescence intensity measurement module.

5. The two-photon fluorescence Z scanning device according to claim 2, characterized in that: The two-photon fluorescence Z scanning device also includes a translation stage, which is used to adjust the positions of the sample to be tested and the fluorescence collection module.

6. A two-photon fluorescence Z scanning measurement method, implemented using the two-photon fluorescence Z scanning device according to any one of claims 1 to 5, characterized in that: The specific steps include: S1: Use a translation stage to move the sample to be tested to the focal position z1 of the focusing mirror, 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 tested, and sends the corresponding collected two-photon fluorescence signal to the fluorescence intensity measurement module; the second energy meter uses the collection mirror to receive the measurement light beam passing through the sample to be tested; 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: Control the sample to be tested to move along the optical axis of the focusing mirror according to a preset step length, and repeat step S1 until h groups of measurement data are obtained, and the wavelength range of the two-photon fluorescence signal of the sample to be tested at different positions is within the wavelength window; the measurement data includes the position z of the sample to be tested, the fluorescence intensity L of the sample to be tested at the current position z, and the pulse energy received by the first energy meter and the second energy meter respectively; S3: The computer completes the measurement of Z scan and two-photon fluorescence data of the sample to be measured based on h groups of measurement data, and the two-photon fluorescence data includes a two-photon absorption coefficient and a two-photon absorption cross section.

7. The two-photon fluorescence Z scanning method according to claim 6, characterized in that: In step S1, the specific steps of obtaining the fluorescence intensity of the sample to be tested at the focal position are: S11: Use the translation stage to move the sample to be tested to the focal position z1 of the focusing mirror; S12: adjusting the position of the fiber optic collimator so that the fluorescence peak intensity of the two-photon fluorescence signal collected by the fiber optic collimator is the highest, and receiving 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 in the wavelength window by the following formula to obtain the fluorescence intensity L1 at the focal position. Otherwise, directly obtain the fluorescence intensity L1 at the focal position by using a photodiode: （1）。 8. The two-photon fluorescence Z-scanning measurement method according to claim 7, characterized in that: The specific steps of step S3 include: S31: The computer calculates the transmittance curve based on the h groups of measurement data Perform fitting; S32: Set the objective function to : （2）； （3）； （4）； in, is the variation of the absorbance of the sample to be tested with position z, is the change of the fluorescence intensity of the sample to be tested with position z, is the logarithm of the ratio of the fluorescence intensity to the absorbance when the sample is at position z. is the logarithm of the ratio of fluorescence intensity to absorbance; S33: From the transmittance curve The expression of : （5）； （6）； （7）； （8）； （9）； in, T 0 is the linear transmittance, λ is the wavelength of the laser pulse, w 0 is the beam waist radius, α is the linear absorption coefficient, is the pulse peak intensity, β is the two-photon absorption coefficient, τ is the pulse width, is the average value of the energy ES1 irradiated to the sample to be tested, , and It is an intermediate parameter for calculating the transmittance curve and has no actual physical meaning; S34: Use the objective function to complete the intermediate parameters Optimization; S33: The optimized intermediate parameters Substituting into equation (8), we obtain the two-photon absorption coefficient β ; S35: Calculate the two-photon absorption cross section of the sample to be tested by the following formula σ (2) : （10）； in, is the photon energy, N 0 is the particle number density.

Images