Method and apparatus for measuring a multiphoton interaction cross section

Abstract

The application provides a kind of multi-photon action cross section measurement method and device, and the measurement method comprises: determining the target excitation mode type of the excitation wavelength to be measured;Wherein, excitation mode type includes any one of the following: three-photon excitation mode and four-photon excitation mode;Based on the target excitation mode type, the target action cross section value ratio formula is obtained;Wherein, the action cross section value ratio formula includes any one of the following: three-photon ratio formula and four-photon ratio formula;The reference excitation wavelength action cross section value of the same excitation mode type is input into the target action cross section value ratio formula, and the action cross section value of the excitation wavelength to be measured is calculated. Adopt fluorescence comparison method, and based on the known action cross section value of 1680nm excitation wavelength, the action cross section value of 2200nm excitation wavelength is obtained, the action cross section measurement of three-photon excitation mode and four-photon excitation mode of 2200nm window fluorescent marker is realized, so as to be able to characterize the multi-photon excitation at 2200nm.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence imaging technology, and particularly relates to a method and device for measuring a multi-photon interaction cross section. BACKGROUND

[0002] Multi-photon microscopy (MPM) is a nonlinear optical imaging technique. Under the irradiation of long-wavelength light with high photon density, a fluorescent molecule can simultaneously absorb two or more photons, jump from a low-energy state to a high-energy state, and then re-jump to a low-energy state in the decay process and emit fluorescence. Multi-photon microscopy can be divided into two-photon microscopy, three-photon microscopy, and four-photon microscopy according to the number of photons that need to be simultaneously absorbed to excite the same fluorescent signal.

[0003] In related technologies, multi-photon excitation wavelengths have evolved through the 800 nm window, the 1300 nm window, and the 1680 nm window. However, there is a lack of related research on the 2200 nm window for deep tissue imaging, and thus the multi-photon excitation at 2200 nm cannot be characterized. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method and device for measuring a multi-photon interaction cross section, aiming to solve the problem that the multi-photon imaging of the 2200 nm window cannot be characterized due to the lack of related research on the multi-photon imaging of the 2200 nm window in related technologies.

[0005] To solve the above technical problem, the present application provides a method for measuring a multi-photon interaction cross section in a first aspect, comprising:

[0006] determining a target excitation mode type of a to-be-measured excitation wavelength; wherein the excitation mode type includes any one of the following: a three-photon excitation mode and a four-photon excitation mode;

[0007] obtaining a target interaction cross section value ratio formula based on the target excitation mode type; wherein the interaction cross section value ratio formula includes any one of the following: a three-photon ratio formula and a four-photon ratio formula;

[0008] inputting a reference excitation wavelength interaction cross section value of the same excitation mode type into the target interaction cross section value ratio formula to calculate the interaction cross section value of the to-be-measured excitation wavelength.

[0009] Preferably, the step of obtaining a target interaction cross section value ratio formula based on the target excitation mode type comprises:

[0010] when the target excitation mode type is a three-photon excitation mode, obtaining the three-photon ratio formula;

[0011] when the target excitation modal type is a four-photon excitation modal, obtaining the four-photon ratio formula.

[0012] Preferably, the three-photon ratio formula is expressed as:

[0013]

[0014] wherein the subscripts 0 and 1 represent the reference sample and the sample to be measured respectively, ησ is the action cross section, P is the power at the focus of the excitation wavelength, τ is the pulse width of the excitation wavelength, S is the fluorescence signal count, C is the molar concentration of the sample, and λ is the excitation wavelength.

[0015] Preferably, the four-photon ratio formula is expressed as:

[0016]

[0017] wherein the subscripts 0 and 1 represent the reference sample and the sample to be measured respectively, ησ is the action cross section, P is the power at the focus of the excitation wavelength, τ is the pulse width of the excitation wavelength, S is the fluorescence signal count, C is the molar concentration of the sample, and λ is the excitation wavelength.

[0018] Preferably, the step of determining the target excitation modal type of the excitation wavelength to be measured comprises:

[0019] based on the one-to-one correspondence between the signal values of the multiple groups of fluorescence signals of the sample and the multiple groups of excitation powers, obtaining a first characteristic representation slope of the excitation wavelength to be measured and a second characteristic representation slope of the reference excitation wavelength;

[0020] determining the target excitation modal type of the excitation wavelength to be measured according to the similarity between the first characteristic representation slope and a preset characteristic representation slope and the similarity size between the second characteristic representation slope and the preset characteristic representation slope.

[0021] Preferably, the step of determining the target excitation modal type of the excitation wavelength to be measured according to the similarity between the first characteristic representation slope and a preset characteristic representation slope and the similarity size between the second characteristic representation slope and the preset characteristic representation slope comprises:

[0022] obtaining a first difference value by subtracting the preset characteristic representation slope from the first characteristic representation slope, and obtaining a second difference value by subtracting the preset characteristic representation slope from the second characteristic representation slope;

[0023] when the preset characteristic representation slope is three and both the first difference value and the second difference value are less than a preset threshold value, determining that the target excitation modal type of the excitation wavelength to be measured is a three-photon excitation modal;

[0024] When the preset characteristic representation slope is four and the first difference value and the second difference value are both less than a preset threshold, it is determined that the target excitation mode type of the to-be-tested excitation wavelength is a four-photon excitation mode.

[0025] Preferably, the one-to-one correspondence between the signal values of the multiple groups of sample-based fluorescence signals and the multiple groups of excitation powers comprises the following steps:

[0026] Linear fitting is performed on the mapping parameters composed of the signal values of the multiple groups of to-be-tested sample fluorescence signals and the excitation powers to obtain a first fitting straight line, and the slope of the first fitting straight line is taken as the first characteristic representation slope of the to-be-tested excitation wavelength.

[0027] Linear fitting is performed on the mapping parameters composed of the signal values of the multiple groups of reference sample fluorescence signals and the excitation powers to obtain a second fitting straight line, and the slope of the second fitting straight line is taken as the second characteristic representation slope of the to-be-tested excitation wavelength.

[0028] The second aspect of the present application also provides a multi-photon interaction cross section measuring device, which adopts the steps in the measuring method of any one of the above to measure the multi-photon interaction cross section, and comprises a laser, a half-wave plate, a polarization beam splitter, a frequency modulation component, a dichroic mirror, an objective lens, a photomultiplier tube, a preamplifier and a photon counter. The laser emitted laser light passes through the half-wave plate, the polarization beam splitter, the frequency modulation component, the dichroic mirror and the objective lens in sequence, and is projected to a to-be-tested sample through the objective lens. Fluorescence collected by the objective lens is back-transmitted to the dichroic mirror, and is reflected by the dichroic mirror to the detection surface of the photomultiplier tube. The preamplifier is electrically connected to the photomultiplier tube, and the photon counter is electrically connected to the preamplifier.

[0029] Preferably, the frequency modulation component comprises a first lens group, a polarization-maintaining large-mode-field fiber, a second lens group and a first filter. The laser emitted laser light passes through the first lens group, the polarization-maintaining large-mode-field fiber, the second lens group and the first filter in sequence.

[0030] Preferably, the multi-photon interaction cross section measuring device further comprises a second filter and a third lens group. The fluorescence reflected by the dichroic mirror is transmitted to the detection surface of the photomultiplier tube after passing through the second filter and the third lens group in sequence.

[0031] Compared with the prior art, the method and device for measuring a multi-photon action cross section have the beneficial effect that: a target excitation mode type of a to-be-measured excitation wavelength is determined; a target action cross section value ratio formula is obtained based on the target excitation mode type; and an action cross section value of the to-be-measured excitation wavelength is calculated by inputting an action cross section value of a reference excitation wavelength of the same excitation mode type into the target action cross section value ratio formula. The fluorescence comparison method is adopted, and the action cross section value of the 2200nm excitation wavelength is obtained based on the known action cross section value of the 1680nm excitation wavelength, so that the action cross section measurement of the three-photon excitation mode and the four-photon excitation mode of the 2200nm window fluorescent marker is realized, and the multi-photon excitation at 2200nm can be characterized. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0033] Figure 1 is the basic flow chart of the multi-photon action cross section measurement method of the embodiment of the present application;

[0034] Figure 2 is the detailed flow chart of the multi-photon action cross section measurement method of the embodiment of the present application;

[0035] Figure 3 is the structural schematic diagram of the multi-photon action cross section measurement device of the embodiment of the present application.

[0036] In the drawings, the reference signs represent: 1, laser; 2, half-wave plate; 3, polarization beam splitter; 4, frequency modulation component; 41, first lens group; 42, polarization maintaining large mode field fiber; 43, second lens group; 44, first filter; 5, dichroic mirror; 6, objective lens; 7, photomultiplier tube; 8, preamplifier; 9, photon counter; 10, second filter; 20, third lens group; 30, to-be-measured sample. DETAILED DESCRIPTION

[0037] The embodiments of the present application will be described in detail below, and the examples of the embodiments are shown in the drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0038] In the description of the present application, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "circumferential", "radial" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, which are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0039] In addition, the terms "first", "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can include one or more of the features explicitly or implicitly. In the description of the present application, the meaning of "multiple" is two or more, unless otherwise specifically limited.

[0040] Embodiment:

[0041] The embodiment of the present application provides a kind of measurement method of multi-photon action cross section, can characterize 2200nm window multi-photon excitation, such as Figure 1 The basic flow chart of the measurement method of multi-photon action cross section provided in the embodiment, the measurement method comprises the following steps:

[0042] Step 101, determine the target excitation mode type of the to-be-tested excitation wavelength.

[0043] Specifically, the to-be-tested excitation wavelength is 2200nm, and the excitation mode type includes any one of three-photon excitation mode and four-photon excitation mode, and the three-photon excitation mode and the four-photon excitation mode have greater signal background ratio and greater theoretical imaging depth.

[0044] Step 102, based on the target excitation mode type, obtain the target action cross section value ratio formula.

[0045] Specifically, by using fluorescence comparison method, the unknown fluorescence marker cross section is calculated by a known cross section fluorescence marker, i.e. the action cross section value ratio formula of the to-be-tested excitation wavelength and the reference excitation wavelength. Different excitation mode types correspond to different action cross section value ratio formulas, specifically: when the to-be-tested excitation wavelength and the reference excitation wavelength are both three-photon excitation mode, then the action cross section value ratio formula is three-photon ratio formula; when the to-be-tested excitation wavelength and the reference excitation wavelength are both four-photon excitation mode, then the action cross section value ratio formula is four-photon ratio formula.

[0046] Step 103, input the reference excitation wavelength of the same excitation mode type into the target action cross-section value ratio formula, and calculate the action cross-section value of the to-be-measured excitation wavelength.

[0047] Specifically, the time-averaged fluorescence photon flux and the incident power of the reference excitation wavelength are input into the preset action cross-section value calculation formula to calculate the action cross-section value of the to-be-measured excitation wavelength, so as to obtain the action cross-section value of the reference excitation wavelength; the action cross-section value calculation formula is represented as:

[0048]

[0049]

[0050] T = 1 / f;

[0051] wherein n is the number of absorbed photons, <F () () is the time-averaged fluorescence photon flux under the multi-photon excitation mode, is the n-order time coherence of the excitation source, f is the laser repetition frequency, τ is the laser pulse width, φ is the system collection efficiency, η is the fluorescence quantum efficiency, σ n is the n-photon absorption cross-section, C is the concentration of the fluorescent probe, n0 is the refractive index of the solution, a2 = 64, a3 = 28.1, a4 = 18.3, NA is the numerical aperture of the objective lens, <P(t)> is the incident power, λ is the excitation wavelength in vacuum; T is the laser pulse period, I0(t) is the intensity at the focal point varying with time.

[0052] It should be noted that the action cross-section value calculation formula is the relationship between the time-averaged fluorescence photon flux and the incident power under two-photon, three-photon and four-photon excitation in the thick sample limit; in the thick sample limit, the generated fluorescence depends on the NA under three-photon and four-photon excitation; is a dimensionless quantity, and for a Gaussian time profile pulse It is assumed that the to-be-measured excitation wavelength has a certain pulse shape, the pulse width τ and and all other quantities in the above action cross-section value calculation formula except the multi-photon action cross-section ησ can be quantitatively measured, so that the action cross-section value of the to-be-measured excitation wavelength can be obtained according to the action cross-section value calculation formula.

[0053] It should be understood that the multi-photon fluorescence signal is proportional to the action cross-section value, that is, the greater the action cross-section value, the higher the signal level, and the deeper the imaging. In the embodiment of the present application, by implementing the above steps, the action cross-section measurement of the three-photon excitation mode and the four-photon excitation mode of the 2200 nm window fluorescence marker is realized by using the fluorescence comparison method and based on the known action cross-section value at the 1680 nm excitation wavelength, so as to be able to characterize the multi-photon excitation at 2200 nm.

[0054] Please refer to Figure 2 , Figure 2 The detailed flowchart of the measurement method of the multi-photon action cross-section provided by the embodiment of the present application, in the embodiment, based on the multi-photon action cross-section of the reference sample under the excitation of the known 1680 nm wavelength (reference excitation wavelength), the multi-photon action cross-section of the to-be-measured sample under the excitation of the 2200 nm wavelength (to-be-measured excitation wavelength) is measured, and the measurement method comprises the following steps:

[0055] Step 201, based on the one-to-one correspondence between the signal values of the multiple groups of fluorescence signals of the sample and the multiple groups of excitation powers, the first characteristic representation slope of the to-be-measured excitation wavelength and the second characteristic representation slope of the reference excitation wavelength are obtained.

[0056] Step 2011: linearly fitting the mapping parameters composed of the signal values of the multiple groups of fluorescence signals of the to-be-measured sample and the excitation powers to obtain a first fitting straight line, and taking the slope of the first fitting straight line as the first characteristic representation slope of the to-be-measured excitation wavelength.

[0057] Specifically, the signal values of the fluorescence signals of the to-be-measured sample are taken as the vertical axis, and the excitation powers are taken as the horizontal axis, and then the mapping parameters are linearly fitted to obtain a fitting straight line. In an embodiment, 0.05 μM fluorescence probe is dissolved in water as the to-be-measured sample (0.05 μM is the concentration of Qtracker800, the sample is sealed in a glass groove, and Qtracker800 is a kind of fluorescence probe), and the first characteristic representation slope of the to-be-measured sample (Qtracker800) is 2.94. In another embodiment, 0.05 μM fluorescence probe is dissolved in water as the to-be-measured sample (0.05 μM is the concentration of Qtracker655, the sample is sealed in a glass groove, and Qtracker655 is a kind of fluorescence probe), and the first characteristic representation slope of the to-be-measured sample (Qtracker655) is 3.81.

[0058] Step 2012: linearly fitting the mapping parameters composed of the signal values of the multiple groups of fluorescence signals of the reference sample and the excitation powers to obtain a second fitting straight line, and taking the slope of the second fitting straight line as the second characteristic representation slope of the to-be-measured excitation wavelength.

[0059] Specifically, the signal value of the reference sample fluorescence signal is taken as the vertical axis, and the excitation power is taken as the horizontal axis, and then the mapping parameters are linearly fitted to obtain a fitting straight line. In an embodiment, 10 μM sulfo rhodamine 101 (SR101 fluorescent dye) is dissolved in phosphate buffered saline as a reference sample of the sample to be tested (Qtracker800), and the slope of the second characteristic of the reference sample (SR101 fluorescent dye) is 2.83. In another embodiment, 100 μM Fluorescein is dissolved in a sodium hydroxide aqueous solution with a pH of 13 as a reference sample of the sample to be tested (Qtracker655), and the slope of the second characteristic of the reference sample (Fluorescein) is 3.85.

[0060] Step 202, the first difference value is obtained by subtracting the preset characteristic slope from the first characteristic slope, and the second difference value is obtained by subtracting the preset characteristic slope from the second characteristic slope.

[0061] Specifically, for a three-photon excitation mode, the preset characteristic slope is three; for a four-photon excitation mode, the preset characteristic slope is four. In an embodiment of the present embodiment, the first characteristic slope of the sample to be tested (Qtracker800) is 2.94, and the corresponding first difference value is 0.06; the second characteristic slope of the reference sample (SR101 fluorescent dye) is 2.83, and the corresponding second difference value is 0.17. In another embodiment of the present embodiment, the first characteristic slope of the sample to be tested (Qtracker655) is 3.81, and the corresponding first difference value is 0.19; the second characteristic slope of the reference sample (Fluorescein) is 3.85, and the corresponding second difference value is 0.15.

[0062] Step 203, when the preset characteristic slope is three and both the first difference value and the second difference value are less than the preset threshold, it is determined that the target excitation mode type of the excitation wavelength to be tested is a three-photon excitation mode; when the preset characteristic slope is four and both the first difference value and the second difference value are less than the preset threshold, it is determined that the target excitation mode type of the excitation wavelength to be tested is a four-photon excitation mode.

[0063] Specifically, the preset threshold is 0.2, and since the first difference and the second difference are both less than the preset threshold, the first characteristic representation slope of the to-be-tested sample (Qtracker800) and the second characteristic representation slope of the reference sample (SR101 fluorescent dye) are both close to the preset characteristic representation slope, that is, the first characteristic representation slope of Qtracker800 excited by the to-be-tested excitation wavelength of 2200 nm is more similar to the second characteristic representation slope of SR101 fluorescent dye excited by the reference excitation wavelength of 1680 nm, thereby verifying that Qtracker800 is excited by three-photon excitation at 2200 nm, that is, the to-be-tested excitation wavelength is a three-photon excitation mode. Similarly, the first characteristic representation slope of the to-be-tested sample (Qtracker655) and the second characteristic representation slope of the reference sample (Fluorescein) are both close to the preset characteristic representation slope, thereby verifying that Qtracker655 is excited by four-photon excitation at 2200 nm, that is, the to-be-tested excitation wavelength is a four-photon excitation mode.

[0064] Step 204, when the target excitation mode type is a three-photon excitation mode, a three-photon ratio formula is obtained; when the target excitation mode type is a four-photon excitation mode, a four-photon ratio formula is obtained.

[0065] Specifically, the three-photon ratio formula is represented as:

[0066]

[0067] wherein the subscripts 0 and 1 respectively represent a reference sample and a to-be-tested sample, η is quantum efficiency, σ is an absorption cross section, P is power at an excitation wavelength focal point, τ is pulse width of the excitation wavelength, S is fluorescent signal count, C is molar concentration of the sample, and λ is the excitation wavelength.

[0068] The four-photon ratio formula is represented as:

[0069]

[0070] wherein the subscripts 0 and 1 respectively represent a reference sample and a to-be-tested sample, η is quantum efficiency, σ is an absorption cross section, P is power at an excitation wavelength focal point, τ is pulse width of the excitation wavelength, S is fluorescent signal count, C is molar concentration of the sample, and λ is the excitation wavelength.

[0071] Step 205, inputting the reference excitation wavelength cross section value of the same excitation mode type into the target cross section value ratio formula to calculate the cross section value of the to-be-tested excitation wavelength.

[0072] In this embodiment, for the measurement of three-photon action cross section, SR101 is excited as a reference sample with a known reference excitation wavelength (1680 nm), the three-photon action cross section of the reference sample SR101 excited at 1680 nm is η0σ0= 65.5 x 10 -84 cm 6 (s / photon) 2 The pulse width at the reference sample SR101 is measured to be τ0= 68 fs using a second-order interferometric autocorrelator, the pulse width at the sample under test Qtracker800 is measured to be τ1= 90 fs, S0and S1are the counts of three-photon fluorescence signals measured at the reference excitation wavelength (1680 nm) and the excitation wavelength under test (2200 nm) respectively, C0and C1are the concentrations of the reference sample SR101 and the sample under test Qtracker800 respectively, finally, the η1σ1of the sample under test Qtracker800 at the 2200 nm window is calculated to be η1σ1= 1.68 x 10 -78 cm 6 (s / photon) 2 , which is 4 orders of magnitude larger than the reference sample SR101 excited at the reference excitation wavelength (1680 nm).

[0073] For the measurement of four-photon action cross section, Fluorescein is excited as a reference sample with a known reference excitation wavelength (1680 nm), the four-photon action cross section of the reference sample Fluorescein excited at 1680 nm is η0σ0= 10.5 x 10 -116 cm 8 (s / photon) 3 The pulse width at the reference sample Fluorescein is measured to be τ0= 68 fs using a second-order interferometric autocorrelator, the pulse width at the sample under test Qtracker655 is measured to be τ1= 90 fs, S0and S1are the counts of four-photon fluorescence signals measured at the reference excitation wavelength (1680 nm) and the excitation wavelength under test (2200 nm) respectively, C0and C1are the concentrations of the reference sample Fluorescein and the sample under test Qtracker655 respectively, finally, the η1σ1of the sample under test Qtracker655 at the 2200 nm window is calculated to be η1σ1= 7.8 x 10 -108 cm 8 (s / photon) 3 , which is 8 orders of magnitude larger than the reference sample Fluorescein excited at the reference excitation wavelength (1680 nm).

[0074] Please refer to Figure 3The embodiment of the present application also provides a multi-photon interaction cross section measuring device which adopts the steps in the measuring method to measure the multi-photon interaction cross section, and the multi-photon interaction cross section measuring device comprises a laser 1, a half-wave plate 2, a polarization beam splitter 3, a frequency modulation assembly 4, a dichroic mirror 5, an objective lens 6, a photomultiplier tube 7, a preamplifier 8 and a photon counter 9, laser emitted by the laser 1 passes through the half-wave plate 2, the polarization beam splitter 3, the frequency modulation assembly 4, the dichroic mirror 5 and the objective lens 6 in sequence, and is projected to a sample to be measured through the objective lens 6, the back transmission of fluorescence collected by the objective lens 6 is reflected to the detection surface of the photomultiplier tube 7 by the dichroic mirror 5, the preamplifier 8 is electrically connected to the photomultiplier tube 7, and the photon counter 9 is electrically connected to the preamplifier 8.

[0075] Specifically, the laser 1 can be a 1550nm wavelength fiber laser, the laser emitted by the laser 1 is changed to 2200nm wavelength in the frequency modulation assembly 4 by using the soliton self-frequency shift (SSFS) technology, so that the fluorescence microscopic imaging of 50um depth on the surface of the sample to be measured is realized; since the laser emitted by the laser 1 is linearly polarized light, the half-wave plate 2 can make the polarization of the laser align with the main axis of the frequency modulation assembly 4; the polarization beam splitter 3 is used for coupling two beams of orthogonal polarized light into the frequency modulation assembly 4; the dichroic mirror 5 can be a 965nm long-wave pass dichroic mirror; the objective lens 6 is an air objective lens 6 with a numerical aperture NA of 0.45 and a magnification of 20 times, the light beam passing through the dichroic mirror 5 fully fills the objective lens 6, so as to obtain an aperture diffraction limited focus point; in order to facilitate the adjustment of the spatial distance between the sample and the objective lens 6, so that the light beam can be focused at 50um in the sample to be measured, the sample to be measured is fixed on a one-dimensional translation stage; the fluorescence signal reflected by the fluorescent marker on the sample to be measured adopts a back detection technology, that is, the objective lens 6 is used to collect the fluorescence, so that the fluorescence is transmitted in the opposite direction (relative to the light beam transmission direction), and is reflected to the detection surface of the photomultiplier tube 7 by the dichroic mirror 5, then the preamplifier 8 is used to convert the current signal into a low-noise and high-gain voltage signal, and then the voltage signal output by the preamplifier 8 is connected to the photon counter 9, that is, the construction of the 2200nm multi-photon excitation spectrum measuring system is completed.

[0076] In one embodiment, the frequency modulation assembly 4 comprises a first lens group 41, a polarization maintaining large mode area fiber 42, a second lens group 43 and a first filter 44, and the laser emitted by the laser 1 passes through the first lens group 41, the polarization maintaining large mode area fiber 42, the second lens group 43 and the first filter 44 in sequence. Specifically, the length of the polarization maintaining large mode area fiber 42 (LMA) can be 80 cm, and the laser pumped by the 1550 nm fiber laser 1 is transmitted into the polarization maintaining large mode area fiber 42 to generate a 2200 nm soliton by using the soliton self-frequency shift (SSFS) technology; the first lens group 41 and the second lens group 43 can be focusing collimating lenses, which can expand and collimate the light beam, and the light beam passing through the second lens group 43 is incident on the first filter 44 at an angle of 45°; the first filter 44 is a long-wave pass filter, which filters the light beam output by the polarization maintaining large mode area fiber 42, so as to obtain a clean optical soliton.

[0077] In one embodiment, the multi-photon interaction cross-section measuring device further comprises a second filter 10 and a third lens group 20, and the fluorescence reflected by the dichroic mirror 5 is transmitted to the detection surface of the photomultiplier tube 7 after passing through the second filter 10 and the third lens group 20 in sequence. Specifically, the second filter 10 can be a short-wave pass filter, and the third lens is used to expand and collimate the fluorescence reflected by the dichroic mirror 5, and the expanded and collimated fluorescence is transmitted to the detection surface of the photomultiplier tube 7.

[0078] The above only describes the preferred embodiments of the present application and should not be used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method of measuring a multiphoton interaction cross section, the method comprising: The method comprises the steps of: obtaining a first characteristic slope of the to-be-detected excitation wavelength and a second characteristic slope of the reference excitation wavelength based on a one-to-one correspondence between signal values of a plurality of groups of fluorescence signals of the sample and a plurality of groups of excitation powers; determining a target excitation modal type of the to-be-detected excitation wavelength according to similarities of the first characteristic slope and a preset characteristic slope and sizes of similarities of the second characteristic slope and the preset characteristic slope, wherein the excitation modal type comprises any one of a three-photon excitation modal and a four-photon excitation modal; obtaining a target action cross-section value ratio formula based on the target excitation modal type, wherein the action cross-section value ratio formula comprises any one of a three-photon ratio formula and a four-photon ratio formula; inputting an action cross-section value of the reference excitation wavelength of the same excitation modal type into the target action cross-section value ratio formula to obtain an action cross-section value of the to-be-detected excitation wavelength.

2. The method of measuring a multi-photon interaction cross section according to claim 1, wherein, The step of obtaining the target action cross-section value ratio formula based on the target excitation modal type comprises: when the target excitation modal type is the three-photon excitation modal, obtaining the three-photon ratio formula; when the target excitation modal type is the four-photon excitation modal, obtaining the four-photon ratio formula.

3. The method of measuring a multi-photon interaction cross section according to claim 2, wherein The three-photon ratio formula is expressed as: wherein subscripts 0 and 1 respectively represent a reference sample and a to-be-detected sample, ησ is an action cross-section, P is a power at a focal point of an excitation wavelength, τ is a pulse width of the excitation wavelength, S is a fluorescence signal count, C is a molar concentration of the sample, and λ is the excitation wavelength.

4. The method of measuring a multi-photon interaction cross section according to claim 2, wherein The four-photon ratio formula is expressed as: wherein subscripts 0 and 1 respectively represent a reference sample and a to-be-detected sample, ησ is an action cross-section, P is a power at a focal point of an excitation wavelength, τ is a pulse width of the excitation wavelength, S is a fluorescence signal count, C is a molar concentration of the sample, and λ is the excitation wavelength.

5. The method of claim 1, wherein the method is used for measuring a multi-photon interaction cross section. The step of determining the target excitation modal type of the to-be-detected excitation wavelength according to the similarities of the first characteristic slope and the preset characteristic slope and the sizes of the similarities of the second characteristic slope and the preset characteristic slope comprises: obtaining a first difference value by subtracting the preset characteristic slope from the first characteristic slope and obtaining a second difference value by subtracting the preset characteristic slope from the second characteristic slope; when the preset characteristic slope is three and the first difference value and the second difference value are both less than a preset threshold, determining that the target excitation modal type of the to-be-detected excitation wavelength is the three-photon excitation modal; when the preset characteristic slope is four and the first difference value and the second difference value are both less than the preset threshold, determining that the target excitation modal type of the to-be-detected excitation wavelength is the four-photon excitation modal.

6. The method of measuring a multi-photon interaction cross section according to claim 1, wherein The step of obtaining the first characteristic slope of the to-be-detected excitation wavelength and the second characteristic slope of the reference excitation wavelength based on a one-to-one correspondence between signal values of a plurality of groups of fluorescence signals of the sample and a plurality of groups of excitation powers comprises: linearly fitting a mapping parameter composed of signal values of a plurality of groups of fluorescence signals of the to-be-detected sample and excitation powers to obtain a first fitting straight line, and taking a slope of the first fitting straight line as the first characteristic slope of the to-be-detected excitation wavelength; A second fitting straight line is obtained by linear fitting of the mapping parameters composed of the signal values of the fluorescence signals of the multiple groups of reference samples and the excitation power, and a slope of the second fitting straight line is taken as a second characteristic representation slope of the to-be-measured excitation wavelength.

7. A multi-photon interaction cross-section measurement apparatus, characterized by, The multiphoton interaction cross section is measured by the steps in the measurement method of any one of claims 1 to 6, and the multiphoton interaction cross section measurement device comprises a laser, a half-wave plate, a polarization beam splitter, a frequency modulation component, a dichroic mirror, an objective lens, a photomultiplier tube, a preamplifier and a photon counter, laser emitted by the laser passes through the half-wave plate, the polarization beam splitter, the frequency modulation component, the dichroic mirror and the objective lens in sequence, and is projected to the to-be-measured sample through the objective lens, the back transmission of the fluorescence collected by the objective lens is transmitted to the dichroic mirror, and is reflected by the dichroic mirror to the detection surface of the photomultiplier tube, the preamplifier is electrically connected to the photomultiplier tube, and the photon counter is electrically connected to the preamplifier.

8. The multi-photon interaction cross section measurement apparatus according to claim 7, wherein The frequency modulation component comprises a first lens group, a polarization maintaining large mode field fiber, a second lens group and a first filter, and the laser emitted by the laser passes through the first lens group, the polarization maintaining large mode field fiber, the second lens group and the first filter in sequence.

9. The multi-photon interaction cross section measurement apparatus according to claim 7, wherein The multiphoton interaction cross section measurement device further comprises a second filter and a third lens group, and the fluorescence reflected by the dichroic mirror is transmitted to the detection surface of the photomultiplier tube after passing through the second filter and the third lens group in sequence.

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