Fluorescent bead multiphoton property measuring device

Abstract

The invention discloses a fluorescent bead multi-photon property measuring device which is characterized in that a laser signal with a preset first wavelength is generated through an optical fiber laser, and a photonic crystal fiber converts the laser signal into a high-energy soliton laser signal with a preset second wavelength (in a target wave band range); then the high-energy soliton laser signal is accurately focused on a fluorescent bead sample through an optical focusing assembly, the fluorescent bead sample is effectively excited to generate a corresponding three-photon fluorescent signal, and finally a microscopic image and a three-photon emission spectrum of the fluorescent bead are obtained at the same time in combination with a microscopic imaging assembly and a spectrograph. Therefore, comprehensive measurement of the multi-photon properties of the fluorescent beads can be efficiently and accurately realized, and the measurement effectiveness and efficiency are improved. Besides, the device can also measure the multi-photon order and the relative multi-photon cross section of the fluorescent bead, and evaluates the photobleaching behavior and the light stability of the fluorescent bead in combination with the dynamic monitoring of the fluorescent signal along with time, thereby making up the defects of the related technology in the measurement of the multi-photon property of the fluorescent bead at the wave band of 1700nm.

Description

Technical Field

[0001] The invention relates to the technical field of optical imaging, and in particular to a device for measuring multi-photon properties of fluorescent beads. Background Art

[0002] Fluorescent beads are tiny particles with fluorescent properties, and their carriers are usually organic or inorganic materials. Fluorescent beads have been widely used in many fields due to their high sensitivity, good luminescence performance, and strong stability. For example, in the fields of particle tracking, biological and food testing, molecular probes, and fluorescent labeling, fluorescent beads can provide high-contrast signals and are a very valuable tool. In the biomedical field, fluorescent beads can be used as cell markers to observe the movement and distribution of cells through fluorescence microscopy or flow cytometry. In addition, due to the small size of fluorescent beads, they are often regarded as ideal point light sources in optical imaging systems. When the diameter of the fluorescent beads is small enough, its intensity distribution function (Beads spread function, BSF) can be approximated as the point spread function (PSF) of the microscope system. Therefore, by imaging the fluorescent beads, the BSF of the focal plane of the microscope objective can be measured to evaluate the resolution of the microscope system.

[0003] Multiphoton fluorescence microscopy (MPM) is a non-invasive nonlinear optical imaging technology with advantages such as subcellular resolution, deep tissue imaging, three-dimensional imaging and dynamic tracking, and has shown great potential in biomedical applications. At present, the multiphoton excitation window for deep tissue imaging mainly includes the following bands: 800nm ​​band (NIR-I, 650-950nm), 1300nm band (NIR-II, 1000-1350nm), 1700nm band (NIR-III, 1600-1840nm) and 2200nm band (NIR-IV, 2100-2300nm). Among them, the 1700nm band has the least light attenuation due to the absorption and scattering of light of this wavelength by tissue, and is considered to be an ideal choice for deep multiphoton imaging. For example, studies have shown that the use of 1700nm band femtosecond pulses can achieve two-photon or three-photon imaging with a depth of more than 2000μm, which is close to the theoretical limit.

[0004] Quantum dot fluorescent beads are nano-fluorescent particles made of quantum dots doped with polymer materials, which have the advantages of high fluorescence intensity and strong photostability. The optical properties of quantum dot fluorescent beads are far superior to traditional fluorophores. The fluorescence intensity of a single quantum dot is several orders of magnitude higher than that of traditional fluorophores (such as rhodamine), and the synergistic effect of multiple quantum dots in the fluorescent beads further enhances the intensity of the fluorescence signal. In addition, the quantum dots are wrapped in a polymer shell, which not only improves the mechanical protection performance, but also effectively isolates the interference of the external environment on the fluorescence performance, further improving the stability of the quantum dot fluorescent beads.

[0005] However, the measurement devices in related technologies are often unable to effectively measure the properties of fluorescent beads in the 1700nm band and maintain high measurement efficiency due to factors such as light source band limitations, detection equipment sensitivity, insufficient wavelength adaptability, optical system structural design, and complex parameter control. Summary of the invention

[0006] The main purpose of the present invention is to propose a device for measuring the multi-photon properties of fluorescent beads, aiming to at least solve the technical problems of insufficient measurement effectiveness and low measurement efficiency when the measuring device in the related art measures the properties of fluorescent beads in the 1700nm band.

[0007] To achieve the above object, the technical solution of the present invention is as follows: In a first aspect of the present invention, a device for measuring multi-photon properties of fluorescent beads is provided, wherein the device comprises a fiber laser, a photonic crystal fiber, an optical focusing component, a spectrometer and a microscopic imaging component; The optical fiber laser is used to generate a laser signal of a preset first wavelength; The photonic crystal fiber is used to convert the laser signal into a preset second wavelength high-energy soliton laser signal; wherein the second wavelength is greater than the first wavelength and is within a target wavelength range; The optical focusing assembly is used to focus the high-energy soliton laser signal onto the fluorescent bead sample and generate a corresponding three-photon fluorescence signal; The spectrometer is used to detect the three-photon fluorescence signal to obtain the three-photon emission spectrum of the fluorescent bead sample; The microscopic imaging component is used to perform microscopic imaging on the three-photon fluorescence signal to obtain a microscopic image of the fluorescent bead sample.

[0008] Based on the first aspect, the fluorescent bead multi-photon property measurement device also includes a spectroscope; the spectroscope is used to split the three-photon fluorescence signal into a first three-photon fluorescence signal and a second three-photon fluorescence signal, and respectively transmit the first three-photon fluorescence signal and the second three-photon fluorescence signal to the spectrometer and the microscopic imaging component.

[0009] Based on the first aspect, the microscopic imaging component includes a photomultiplier tube, a long-pass filter and a microscopic imaging module; the long-pass filter is arranged at the input end of the photomultiplier tube and is used to filter the first three-photon fluorescence signal; the photomultiplier tube is used to detect the intensity of the first three-photon fluorescence signal after filtering; the microscopic imaging module is used to output a microscopic image of the fluorescent bead sample according to the intensity of the first three-photon fluorescence signal.

[0010] Based on the first aspect, the variable neutral density attenuation plate is used to adjust the excitation light power, the microscopic imaging module is used to generate multiple microscopic images corresponding to different excitation light powers, and the relationship curve between the intensity of the first three-photon fluorescence signal and the excitation light power is fit in a pre-constructed logarithmic coordinate system according to the multiple microscopic images, and the output includes the three-photon absorption characteristics of the relationship curve.

[0011] On the basis of the first aspect, the microscopic imaging module further integrates a relative multi-photon cross-section calculation module; the relative multi-photon cross-section calculation module obtains the intensity of the first three-photon fluorescence signal and the excitation light power, and calculates the relative multi-photon cross-section of the fluorescent bead sample using a preset relationship; wherein the relationship is , represents the relative multiphoton cross section, represents the intensity of the first three-photon fluorescence signal, represents the excitation light power.

[0012] Based on the first aspect, the microscopic imaging module also integrates a photostability measurement module; when the fiber laser continuously irradiates the fluorescent bead sample and the surface power of the fluorescent bead sample reaches a preset power range; the photostability measurement module is used to collect the intensity change of the first three-photon fluorescence signal over time, and output a photostability analysis result; wherein the photostability analysis result includes a relationship curve between the irradiation time and the normalized fluorescence intensity.

[0013] Based on the first aspect, the fluorescent bead multi-photon property measurement device also includes a multimode optical fiber, and the spectrometer includes an EMCCD spectrometer configured with a 150-line grating; the multimode optical fiber is used to couple the second three-photon fluorescence signal to the EMCCD spectrometer; the EMCCD spectrometer is used to measure the three-photon emission spectrum of the second three-photon fluorescence signal through the 150-line grating under the condition that the exposure time is a preset number of seconds; wherein the three-photon emission spectrum includes the relationship between the normalized intensity and wavelength of the second three-photon fluorescence signal.

[0014] On the basis of the first aspect, the types of fluorescent bead samples include carboxyl-modified fluorescent bead samples and polystyrene quantum dot fluorescent bead samples; the spectrometer and the image acquisition component are used to process the two-way three-photon fluorescence signals of the carboxyl-modified fluorescent bead samples in the first measurement stage, respectively, to obtain the multi-photon emission spectrum and microscopic image of the carboxyl-modified fluorescent beads in the carboxyl-modified fluorescent bead samples; the spectrometer and the image acquisition component are also used to process the two-way three-photon fluorescence signals of the polystyrene quantum dot fluorescent bead samples in the second measurement stage, respectively, to obtain the multi-photon emission spectrum and microscopic image of the polystyrene quantum dot fluorescent beads in the polystyrene quantum dot fluorescent bead samples.

[0015] Based on the first aspect, the structure of the fluorescent bead sample includes an agar base, a glass slide and fluorescent beads, the agar base is formed by cooling and solidifying a 0.2% agar solution, the fluorescent beads are dispersed in the agar base, the ratio of the fluorescent beads to the agar solution is 1:400, and the agar base is located on the surface of the glass slide; the fluorescent beads are carboxyl-modified fluorescent beads or polystyrene quantum dot fluorescent beads.

[0016] Based on the first aspect, the optical focusing assembly includes a scanning galvanometer, a scanning lens, a sleeve lens and a coated objective lens; the scanning galvanometer is used to control the transmission of the high-energy soliton laser signal according to a preset scanning path; the scanning lens is used to perform a first-stage focusing on the high-energy soliton laser signal in the scanning path, and expand the high-energy soliton laser signal to a spot ratio of 1:4 together with the sleeve lens; the coated objective lens is used to perform a second-stage focusing on the expanded high-energy soliton laser signal to focus on the fluorescent bead sample and generate a three-photon fluorescence signal.

[0017] The fluorescent bead multi-photon property measurement device of the present invention first generates a laser signal of a preset first wavelength (not belonging to the target band range) through an optical fiber laser, and the photonic crystal fiber converts the laser signal into a preset second wavelength (in the target band range) high-energy soliton laser signal, and then accurately focuses the high-energy soliton laser signal on the fluorescent bead sample through an optical focusing component, effectively stimulating it to generate a corresponding three-photon fluorescence signal, and finally combines the microscopic imaging component and the spectrometer to simultaneously obtain the microscopic image and three-photon emission spectrum of the fluorescent bead, so as to achieve a comprehensive measurement of the multi-photon properties of the fluorescent bead efficiently and accurately, and improve the measurement effectiveness and efficiency. In addition, by adjusting the excitation light power, the device can also measure the multi-photon order and relative multi-photon cross-section of the fluorescent bead, and combine the dynamic monitoring of the fluorescence signal over time to evaluate the photobleaching behavior and photostability of the fluorescent bead. It can be seen that the measurement device not only makes up for the shortcomings of the relevant technology in the measurement of the multi-photon properties of fluorescent beads in the 1700nm band, but also provides a scientific basis and technical support for the practical application of fluorescent beads in the fields of deep tissue imaging and particle tracking. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 A schematic diagram of the structure of a fluorescent bead multiphoton property measurement device provided in an embodiment of the present application; Figure 2 A schematic diagram of the structure of a fluorescent bead multiphoton property measurement device provided in an embodiment of the present application; Figure 3 This is a spectrum diagram of a high-energy soliton laser signal in an embodiment of the present application; Figure 4 This is a microscopic image of carboxyl-modified fluorescent beads in the examples of this application; Figure 5 This is a microscopic image of polystyrene quantum dot fluorescent beads in the examples of this application; Figure 6 The relationship curve between the three-photon fluorescence signal and power of the carboxyl-modified fluorescent beads in the examples of this application; Figure 7 The relationship curve between the three-photon fluorescence signal and power of the polystyrene quantum dot fluorescent beads in the embodiment of the present application; Figure 8 The three-photon emission spectrum of the carboxyl-modified fluorescent beads of the present application example; Fig. 9The three-photon emission spectrum of the polystyrene quantum dot fluorescent beads of the present application example; Fig.10 This is a curve showing the relationship between the irradiation time and the normalized fluorescence intensity of the carboxyl-modified fluorescent beads and the polystyrene quantum dot fluorescent beads in the examples of the present application.

[0020] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0021] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0022] It should be noted that related terms such as "first", "second", etc. can be used to describe various components, but these terms do not limit the components. These terms are only used to distinguish one component from another component. For example, without departing from the scope of the present invention, a first component can be referred to as a second component, and the second component can also be referred to as a first component similarly. The term "and / or" refers to any one or more combinations of related items and description items.

[0023] See also Figure 1 and Figure 2 The fluorescent bead multi-photon property measurement device of the embodiment of the present application includes a fiber laser 10, a photonic crystal fiber 20, an optical focusing component 30, a spectrometer 40 and a microscopic imaging component 50.

[0024] In the fluorescent bead multi-photon property measurement device, the electrical connections of each device are as follows: the output end of the fiber laser 10 is connected to the input end of the photonic crystal fiber 20, the output end of the photonic crystal fiber 20 is connected to the input end of the optical focusing component 30, and the output end of the optical focusing component 30 is connected to the input end of the spectrometer 40 and the input end of the microscopic imaging component 50 respectively.

[0025] The fiber laser 10 may be a pump laser with a repetition frequency of 1 MHz and a pulse width of 500 fs, which is used to generate a laser signal with a first wavelength (the wavelength may be 1500-1550 nm) and transmit it to the photonic crystal fiber 20 .

[0026] The photonic crystal fiber 20 may be a rod-shaped photonic crystal fiber with a length of 44 cm and a core diameter of 100 μm. The rod-shaped photonic crystal fiber converts a laser signal of a first wavelength (not belonging to the target wavelength range) into a high-energy soliton laser signal of a preset second wavelength (belonging to the target wavelength range, such as the 1700 nm wavelength range) based on the soliton self-frequency shift effect, and transmits it to the optical focusing component 30. Specifically, the photonic crystal fiber uses its special waveguide structure to broaden the laser signal of the first wavelength through a nonlinear effect and generate a soliton pulse signal of the second wavelength (spectrum such as Figure 3 The optimized fiber parameters ensure that the second wavelength of the high-energy soliton laser signal is stable at 1680nm, while having sufficient pulse energy to meet the excitation requirements of the subsequent three-photon fluorescence signal.

[0027] The optical focusing component 30 can be a combination of lenses, galvanometers and other mirrors, and is mainly used to accurately focus the high-energy soliton laser signal of the second wavelength onto the fluorescent bead sample. By optimizing the numerical aperture and focal position of the focusing lens, it is ensured that the high-energy soliton laser signal forms a stable light spot in the target area of ​​the fluorescent bead sample, thereby effectively exciting the fluorescent beads to generate the corresponding three-photon fluorescence signal.

[0028] The spectrometer 40 may be an EMCCD spectrometer (Electron Multiplying Charge Coupled Device), which is used to detect the three-photon fluorescence signal generated by the fluorescent bead sample. That is, the EMCCD spectrometer distinguishes and records the fluorescence signal through a spectroscopic element and a high-sensitivity detector, thereby obtaining the three-photon emission spectrum corresponding to the fluorescent bead sample in the 1700nm band.

[0029] The microscopic imaging component 50 may be a microscopic imaging component including a photomultiplier tube 501, and the microscopic imaging component is controlled by the corresponding control software Scanlmage to perform microscopic imaging on the three-photon fluorescence signal of the fluorescent bead sample. By synchronously triggering with the fiber laser 10, high precision and high efficiency of microscopic imaging are achieved, and then the corresponding microscopic image of the fluorescent bead sample within the 1700nm band is obtained.

[0030] The fluorescent bead multi-photon property measurement device of the present invention first generates a laser signal of a preset first wavelength (not belonging to the target band range) through an optical fiber laser, and the photonic crystal fiber converts the laser signal into a preset second wavelength (in the target band range) high-energy soliton laser signal, and then accurately focuses the high-energy soliton laser signal on the fluorescent bead sample through an optical focusing component, effectively stimulating it to generate a corresponding three-photon fluorescence signal, and finally combines the microscopic imaging component and the spectrometer to simultaneously obtain the microscopic image and three-photon emission spectrum of the fluorescent bead, so as to achieve a comprehensive measurement of the multi-photon properties of the fluorescent bead efficiently and accurately, and improve the measurement effectiveness and efficiency. In addition, by adjusting the excitation light power, the device can also measure the multi-photon order and relative multi-photon cross-section of the fluorescent bead, and combine the dynamic monitoring of the fluorescence signal over time to evaluate the photobleaching behavior and photostability of the fluorescent bead. It can be seen that the measurement device not only makes up for the shortcomings of the relevant technology in the measurement of the multi-photon properties of fluorescent beads in the 1700nm band, but also provides a scientific basis and technical support for the practical application of fluorescent beads in the fields of deep tissue imaging and particle tracking.

[0031] It should be noted that the fluorescent bead multi-photon property measurement device of the embodiment of the present application can measure more than two types of fluorescent beads, and the types of fluorescent bead samples targeted include at least carboxyl-modified fluorescent bead samples and polystyrene quantum dot fluorescent bead samples.

[0032] Specifically, the spectrometer and the image acquisition component are used to process the two-way three-photon fluorescence signals of the carboxyl-modified fluorescent bead sample in the first measurement stage, respectively, to obtain the multi-photon emission spectrum and microscopic image of the carboxyl-modified fluorescent beads in the carboxyl-modified fluorescent bead sample; the spectrometer and the image acquisition component are also used to process the two-way three-photon fluorescence signals of the polystyrene quantum dot fluorescent bead sample in the second measurement stage, respectively, to obtain the multi-photon emission spectrum and microscopic image of the polystyrene quantum dot fluorescent beads in the polystyrene quantum dot fluorescent bead sample.

[0033] In each fluorescent bead sample, the corresponding structure includes an agar base, a glass slide and fluorescent beads. The agar base is formed by cooling and solidifying a 0.2% agar solution. The fluorescent beads are dispersed in the agar base. The ratio of fluorescent beads to agar solution is 1:400. The agar base is located on the surface of the glass slide. The difference between different types of fluorescent bead samples lies in the different fluorescent beads themselves. For example, the fluorescent beads can be carboxyl-modified fluorescent beads or polystyrene quantum dot fluorescent beads.

[0034] When preparing the fluorescent bead sample, 200 mg of agar powder was weighed with an electronic balance and added to 10 mL of pure water. The agar powder and pure water were fully mixed with an experimental vortex mixer and then heated to about 60°C to melt to prepare a 0.2% agar solution. Two types of fluorescent beads, carboxyl-modified fluorescent beads (emission wavelength: 605 nm) and polystyrene quantum dot fluorescent beads (emission wavelength: 680 nm) with a diameter of 1 μm, were dispersed in the agar solution respectively. The ratio of each fluorescent bead to the agar solution was 1:400. During the dispersion process, the solution temperature was controlled to avoid agar solidification. Subsequently, the mixer was used to oscillate so that the fluorescent beads were evenly dispersed in the agar solution. The mixed solution of fluorescent beads and agar was transferred to a glass slide by a pipette for sealing. After sealing, it was placed in a 4°C refrigerator to cool until the agar was completely solidified. Finally, the carboxyl-modified fluorescent bead sample and the polystyrene quantum dot fluorescent bead sample were obtained.

[0035] In an optional implementation of the present embodiment, the fluorescent bead multi-photon property measurement device also includes a spectroscope 70, which is arranged between the optical focusing component 30 and the spectrometer 40 and the microscopic imaging component 50, and is used to divide the three-photon fluorescence signal generated by the fluorescent bead sample into two paths (a first three-photon fluorescence signal and a second three-photon fluorescence signal).

[0036] Specifically, the spectroscope 70 transmits the first three-photon fluorescence signal to the spectrometer 40 in a direction through the beam splitting function for spectral detection; at the same time, the second three-photon fluorescence signal is transmitted to the microscopic imaging component 50 for microscopic imaging. Therefore, by introducing the spectroscope, the synchronous separation and transmission of the fluorescence signal can be achieved, thereby meeting the needs of spectral detection and microscopic imaging at the same time. In addition, the splitting ratio of the spectroscope can be adjusted according to the experimental requirements to ensure that the intensity of the spectral detection and microscopic imaging signals reaches the optimal level. For example, the coating structure of the spectroscope can be adjusted to change its transmittance (T) and reflectivity (R), and the distribution ratio of the light signal can be optimized to meet the specific needs of different experimental scenarios for signal intensity.

[0037] Among them, the relationship between the three-photon fluorescence signal (S) and the power (P) is: .

[0038] When using a 50 / 50 (T:R=50%:50%) spectroscope, the power of the first and second channels after the spectroscope is the same, and the three-photon fluorescence signal is the same; if a 90 / 10 (T:R=90%:10%) spectroscope is used instead, the power of the first channel after the spectroscope is 1.8 times the original, and the three-photon fluorescence signal of the first channel is enhanced to 5.8 times the original. By optimizing the distribution ratio of the light signal, the emission spectrum or microscopic image measurement results can be selectively improved to meet the needs of different experimental scenarios.

[0039] In an optional implementation of this embodiment, the optical focusing assembly includes a scanning galvanometer 302 , a scanning lens 303 , a tube lens 304 and a coated objective lens 130 .

[0040] Specifically, the scanning galvanometer 302 is used to control the high-energy soliton laser signal to be transmitted according to a preset scanning path; the scanning lens 303 is used to perform the first-stage focusing of the high-energy soliton laser signal in the scanning path, and expand the high-energy soliton laser signal to a spot ratio of 1:4 with the sleeve lens 304; the coated objective lens 130 is used to perform the second-stage focusing of the expanded high-energy soliton laser signal to focus on the fluorescent bead sample and generate a three-photon fluorescence signal. That is, in this embodiment, the precise control and focusing of the high-energy soliton laser signal is achieved through the coordinated work of the scanning galvanometer 302, the scanning lens 303, the sleeve lens 304 and the coated objective lens 130.

[0041] In an optional implementation of this embodiment, the microscopic imaging component 50 includes a photomultiplier tube 501, a long-pass filter and a microscopic imaging module. The long-pass filter is arranged at the input end of the photomultiplier tube and is used to filter the first three-photon fluorescence signal to effectively block the short-wave interference signal and only allow the target fluorescence signal to pass, thereby improving the purity and accuracy of signal detection. The photomultiplier tube is used to detect the intensity of the first three-photon fluorescence signal after filtering and generate an electrical signal output corresponding to the intensity. The microscopic imaging module is used to output a microscopic image of the fluorescent bead sample according to the intensity of the first three-photon fluorescence signal (the electrical signal corresponding to the intensity), thereby achieving a high-resolution fluorescence imaging effect, which is helpful for the accurate analysis and observation of the multi-photon properties of the fluorescent beads.

[0042] In an optional implementation of this embodiment, the microscopic imaging module is integrated with a variable neutral density attenuation sheet, which controls the excitation light power entering the fluorescent bead sample by changing its optical density through precise dynamic regulation of the excitation light power and according to the preset light intensity regulation requirements, so that the microscopic imaging module can image the two fluorescent beads (carboxyl-modified fluorescent bead sample and polystyrene quantum dot fluorescent bead sample) under different power conditions, and obtain corresponding microscopic images ( Figure 4 and Figure 5 As shown in Figure 2, the fluorescent bead properties can be measured under multiple power conditions.

[0043] When the two fluorescent beads are actually operated, the microscopic imaging module is used to generate multiple microscopic images corresponding to different excitation light powers for the two fluorescent beads (carboxyl-modified fluorescent beads sample and polystyrene quantum dot fluorescent beads sample), and fit the relationship curve between the intensity of the first three-photon fluorescence signal and the excitation light power in the pre-constructed logarithmic coordinate system according to the multiple microscopic images (the relationship curves corresponding to the two different fluorescent beads are shown in FIG. Figure 6 and Figure 7 As shown in the figure, the linear fitting slopes of carboxyl-modified fluorescent beads and polystyrene quantum dot fluorescent beads at 1680nm wavelength are 3.14 and 3.07 respectively, indicating that both fluorescent beads absorb three-photon at 1680nm wavelength and produce three-photon fluorescence. That is, the microscopic imaging module can finally obtain the three-photon absorption characteristics corresponding to the two different fluorescent beads, including the relationship curve.

[0044] In an optional implementation of this embodiment, the microscopic imaging module further integrates a relative multiphoton cross-section calculation module, which obtains the intensity and excitation light power of the first three-photon fluorescence signal and calculates the relative multiphoton cross-section value of the fluorescent bead sample using a preset relationship; wherein the relationship is: , represents the relative multiphoton cross-section value, represents the intensity of the first three-photon fluorescence signal, Indicates the excitation light power.

[0045] When actually operating the two fluorescent beads, the multiphoton cross-section calculation module automatically completes the curve fitting between the three-photon fluorescence signal and the excitation light power based on multiple sets of excitation power and fluorescence intensity data, and calculates the relative action cross-section values ​​of the carboxyl-modified fluorescent beads and polystyrene quantum dot fluorescent beads as 2202.37 and 664.26, respectively, according to the above formula. It can be seen that through the calculation of the relative action cross-section value, the microscopic imaging module can provide quantitative characterization of the multiphoton absorption capacity under different experimental conditions, further enhancing the practicality and accuracy of the device in the study of multiphoton properties.

[0046] In an optional implementation of this embodiment, the fluorescent bead multiphoton property measurement device further includes a multimode optical fiber 80, and the spectrometer includes an EMCCD spectrometer configured with a 150-line grating, the blazing wavelength of the 150-line grating is 800nm, and the minimum spectral resolution is 1.5nm. When measuring the emission spectrum, the multimode optical fiber 80 is used to couple the second three-photon fluorescence signal to the EMCCD spectrometer, and the EMCCD spectrometer is used to measure the three-photon emission spectrum (such as the emission spectrum of the second three-photon fluorescence signal) through the 150-line grating under the condition of an exposure time of 20 seconds. Figure 8 , Fig. 9 Three-photon emission spectra corresponding to different fluorescent beads shown); wherein the three-photon emission spectrum includes the relationship between the normalized intensity and wavelength of the second three-photon fluorescence signal.

[0047] In the specific measurement process, the EMCCD spectrometer performs spectral separation and detection of the second three-photon fluorescence signal through a 150-line grating under the condition of an exposure time of a preset number of seconds (for example, 20 seconds), and records the spectral distribution of the fluorescence intensity as the wavelength changes. The measurement results are output in the form of a three-photon emission spectrum, where the three-photon emission spectrum shows the relationship between the normalized intensity and wavelength of the second three-photon fluorescence signal, which can be used to characterize the spectral characteristics and multi-photon luminescence behavior of the fluorescent beads. This design scheme can achieve high-sensitivity three-photon emission spectrum measurement, which is helpful for analyzing the luminescence mechanism and spectral characteristics of fluorescent beads under three-photon excitation conditions, and further expands the application potential of fluorescent probes in biological imaging, materials science and optical research.

[0048] In an optional implementation of this embodiment, the microscopic imaging module is further integrated with a photostability measurement module. When the optical fiber laser continuously irradiates the fluorescent bead sample for 5 minutes, and the scanning field of view is magnified 30 times and the surface power of the fluorescent bead sample reaches a preset power value (for example, 4mw), the photostability measurement module is used to collect the intensity change of the first three-photon fluorescence signal over time and output the photostability analysis result; wherein the photostability analysis result includes a relationship curve between the irradiation time and the normalized fluorescence intensity (such as Fig. 9 As shown). It can be seen that after 5 minutes of laser irradiation, the three-photon fluorescence intensity of carboxyl-modified fluorescent beads and polystyrene quantum dot fluorescent beads dropped to 87% and 58% of the original intensity, respectively. This shows that the carboxyl-modified fluorescent beads have better photostability. In contrast, the polystyrene quantum dot fluorescent beads show more obvious light attenuation.

[0049] Specifically, the photostability measurement module detects the intensity of the fluorescence signal through a high-precision photomultiplier tube, and with the cooperation of a long-pass filter, ensures that only three-photon fluorescence signals are recorded to avoid the influence of short-wave interference. After data collection is completed, the photostability measurement module generates a curve of the relationship between the normalized fluorescence intensity and the irradiation time based on the recorded data of the fluorescence signal intensity changing over time, so as to intuitively display the photostability performance of the fluorescent beads.

[0050] It can be seen that the photostability analysis results are used to evaluate the photobleaching behavior of fluorescent bead samples under specific excitation conditions, and further determine the degree of signal attenuation of fluorescent beads under long-term excitation, providing quantitative photostability data support for the screening and optimization of fluorescent materials, which is helpful to select fluorescent probe materials with better photostability in biological imaging and optical labeling applications.

[0051] Further analysis based on microscopic images, relative action cross sections and photostability showed that carboxyl-modified fluorescent beads were superior to polystyrene quantum dot fluorescent beads in terms of relative action cross sections and stability. Specifically, the relative three-photon action cross section of the former is 3.3 times that of the latter; after five minutes of continuous irradiation, the three-photon fluorescence signals of the former and the latter decayed by 13% and 42%, respectively. The comprehensive analysis of the performance of fluorescent beads (including brightness and attenuation) provides a scientific basis for the selection of target fluorescent beads. In addition, the measurement of the multiphoton emission spectrum further completes the characterization of the multiphoton emission properties of fluorescent beads.

[0052] Please refer again Figure 2 The fluorescent bead multi-photon property measurement device also includes a focusing lens 90, a collimating lens 110, a long-wave pass filter 120, a reflector 301, a reflector 305 and a dichroic film 60.

[0053] The focusing lens 90 is disposed between the fiber laser 10 and the photonic crystal fiber 20, and is used to converge the 1550nm laser signal generated by the fiber laser 10, thereby ensuring that the laser can be effectively coupled into the photonic crystal fiber 20. Its optical properties can significantly affect the diameter and numerical aperture of the beam entering the optical fiber, thereby affecting the soliton generation efficiency and laser conversion quality in the photonic crystal fiber 20.

[0054] The collimating lens 110 is installed at the output end of the photonic crystal fiber 20. Its function is to collimate the laser signal diverging in the optical fiber to form a parallel light beam so that the laser signal in the subsequent optical path can continue to propagate with a stable direction and beam quality, which helps to improve the transmission efficiency and focusing accuracy of the optical system.

[0055] The long-wave filter 120 is installed at the output end of the photonic crystal fiber 20 to perform wavelength selective filtering on the output optical signal. The filter can block short-wavelength pump light and only allow high-energy soliton laser signals with wavelengths greater than a set threshold (such as 1680nm) to pass, thereby effectively eliminating residual pump light interference and ensuring that the excitation light wavelength matches the experimental requirements.

[0056] The reflector 301 can be selectively arranged at the output end of the long-wave pass filter 120 to change the direction of the laser light path and reflect the high-energy soliton laser signal filtered by the long-wave pass filter 120 to the XY scanning galvanometer 302, so as to facilitate the subsequent beam scanning and sample excitation. Its high reflectivity surface can effectively reduce light loss and ensure the energy transmission stability of the excitation light.

[0057] The reflector 305 is disposed at the output end of the sleeve lens 304, and its high reflectivity ensures that the high-energy soliton laser signal can be accurately guided to the dichroic plate 60 with the lowest possible loss, thereby achieving the direction control of the excitation light and the subsequent optical path separation function.

[0058] The dichroic filter 60 is arranged at the output end of the reflector 305, and is used to separate the excitation light and the three-photon fluorescence signal. The principle is based on the high reflection and high transmission characteristics of a specific wavelength band. When the excitation light is incident, the dichroic filter can transmit the excitation light to the fluorescent bead sample area; and when the fluorescence signal returns from the sample, the dichroic filter will reflect the long-wave fluorescence signal to the detection channel, thereby achieving effective separation of the excitation light and the fluorescence signal, and avoiding the interference of the excitation light with the fluorescence detection.

[0059] In an optional implementation of this embodiment, the fluorescent bead multiphoton property measurement device also includes a host computer, which is used to receive three-photon emission spectra and microscopic images of different types of fluorescent bead samples transmitted by the fluorescent bead multiphoton property measurement device for display and difference analysis.

[0060] The fluorescent bead multi-photon property measurement device of the present invention first generates a high-energy soliton laser signal with a wavelength of 1680nm (belonging to the 1700nm band range) through a fiber laser and a photonic crystal fiber, and then accurately focuses the high-energy soliton laser signal on the fluorescent bead sample through an optical focusing component, effectively stimulating it to generate a corresponding three-photon fluorescence signal, and finally combines the microscopic imaging component and the spectrometer to simultaneously obtain the microscopic image and three-photon emission spectrum of the fluorescent beads, thereby being able to efficiently and accurately achieve a comprehensive measurement of the multi-photon properties of the fluorescent beads, and improve the effectiveness and efficiency of the measurement. In addition, by adjusting the excitation light power, the device can also measure the multi-photon order and relative multi-photon cross-section of the fluorescent beads, and combine the dynamic monitoring of the fluorescence signal over time to evaluate the photobleaching behavior and photostability of the fluorescent beads. It can be seen that the measurement device not only makes up for the shortcomings of the relevant technology in the measurement of the multi-photon properties of fluorescent beads in the 1700nm band, but also provides a scientific basis and technical support for the practical application of fluorescent beads in the fields of deep tissue imaging and particle tracking.

[0061] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0062] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A fluorescent bead multiphoton property measurement device, characterized in that: The fluorescent bead multi-photon property measurement device comprises a fiber laser, a photonic crystal fiber, an optical focusing component, a spectrometer and a microscopic imaging component; The optical fiber laser is used to generate a laser signal of a preset first wavelength; The photonic crystal fiber is used to convert the laser signal into a high-energy soliton laser signal of a preset second wavelength; wherein the second wavelength is greater than the first wavelength and is within a target wavelength range; The optical focusing assembly is used to focus the high-energy soliton laser signal onto the fluorescent bead sample and generate a corresponding three-photon fluorescence signal; The spectrometer is used to detect the three-photon fluorescence signal to obtain the three-photon emission spectrum of the fluorescent bead sample; The microscopic imaging component is used to perform microscopic imaging on the three-photon fluorescence signal to obtain a microscopic image of the fluorescent bead sample.

2. The fluorescent bead multiphoton property measurement device according to claim 1, characterized in that: The fluorescent bead multi-photon property measurement device also includes a spectroscope; The spectroscope is used to split the three-photon fluorescence signal into a first three-photon fluorescence signal and a second three-photon fluorescence signal, and transmit the first three-photon fluorescence signal and the second three-photon fluorescence signal to the spectrometer and the microscopic imaging component respectively.

3. The fluorescent bead multiphoton property measurement device according to claim 2, characterized in that: The microscopic imaging assembly includes a photomultiplier tube, a long-pass filter and a microscopic imaging module; The long-pass filter is arranged at the input end of the photomultiplier tube and is used to filter the first three-photon fluorescence signal; The photomultiplier tube is used to detect the intensity of the first three-photon fluorescence signal after filtering; The microscopic imaging module is used to output a microscopic image of the fluorescent bead sample according to the intensity of the first three-photon fluorescence signal.

4. The fluorescent bead multiphoton property measurement device according to claim 3, characterized in that: The microscopic imaging module is integrated with a variable medium density attenuation sheet, and the variable medium density attenuation sheet is used to adjust the excitation light power; The microscopic imaging module is used to generate multiple microscopic images corresponding to different excitation light powers, and fit the relationship curve between the intensity of the first three-photon fluorescence signal and the excitation light power in a pre-constructed logarithmic coordinate system based on the multiple microscopic images, and output the three-photon absorption characteristics including the relationship curve.

5. The fluorescent bead multiphoton property measurement device according to claim 4, characterized in that: The microscopic imaging module also integrates a relative multi-photon cross-section calculation module; The relative multi-photon cross section calculation module obtains the intensity of the first three-photon fluorescence signal and the excitation light power, and calculates the relative multi-photon cross section of the fluorescent bead sample using a preset relationship; Among them, the relationship is , represents the relative multiphoton cross section, represents the intensity of the first three-photon fluorescence signal, represents the excitation light power.

6. The fluorescent bead multiphoton property measurement device according to claim 4, characterized in that: The microscopic imaging module also integrates a photostability measurement module; When the optical fiber laser continuously irradiates the fluorescent bead sample and the surface power of the fluorescent bead sample reaches a preset power range; The photostability measurement module is used to collect the intensity change of the first three-photon fluorescence signal over time and output a photostability analysis result; wherein the photostability analysis result includes a relationship curve between the irradiation time and the normalized fluorescence intensity.

7. The fluorescent bead multiphoton property measurement device according to claim 2, characterized in that: The fluorescent bead multi-photon property measurement device further comprises a multimode optical fiber, and the spectrometer comprises an EMCCD spectrometer configured with a 150-line grating; The multimode optical fiber is used to couple the second three-photon fluorescence signal to the EMCCD spectrometer; The EMCCD spectrometer is used to measure the three-photon emission spectrum of the second three-photon fluorescence signal through the 150-line grating under the condition of an exposure time of a preset number of seconds; wherein the three-photon emission spectrum includes the relationship between the normalized intensity and wavelength of the second three-photon fluorescence signal.

8. The fluorescent bead multiphoton property measurement device according to claim 2, characterized in that: The types of fluorescent bead samples include carboxyl-modified fluorescent bead samples and polystyrene quantum dot fluorescent bead samples; The spectrometer and the image acquisition component are used to process two three-photon fluorescence signals of the carboxyl-modified fluorescent bead sample in a first measurement phase, respectively, to obtain a multi-photon emission spectrum and a microscopic image of the carboxyl-modified fluorescent beads in the carboxyl-modified fluorescent bead sample; The spectrometer and the image acquisition component are also used to process the two-way three-photon fluorescence signals of the polystyrene quantum dot fluorescent bead sample in the second measurement phase to obtain the multi-photon emission spectrum and microscopic image of the polystyrene quantum dot fluorescent beads in the polystyrene quantum dot fluorescent bead sample.

9. The fluorescent bead multiphoton property measurement device according to claim 8, characterized in that: The structure of the fluorescent bead sample includes an agar base, a glass slide and fluorescent beads, the agar base is formed by cooling and solidifying a 0.2% agar solution, the fluorescent beads are dispersed in the agar base, the ratio of the fluorescent beads to the agar solution is 1:400, and the agar base is located on the surface of the glass slide; The fluorescent beads are carboxyl-modified fluorescent beads or polystyrene quantum dot fluorescent beads.

10. The fluorescent bead multiphoton property measurement device according to claim 1, characterized in that: The optical focusing assembly includes a scanning galvanometer, a scanning lens, a sleeve lens and a coated objective lens; The scanning galvanometer is used to control the high-energy soliton laser signal to be transmitted according to a preset scanning path; The scanning lens is used to perform a first-stage focusing on the high-energy soliton laser signal in the scanning path, and expand the high-energy soliton laser signal to a spot ratio of 1:4 together with the sleeve lens; The coated objective lens is used to perform a second-stage focusing on the expanded high-energy soliton laser signal so as to focus it onto the fluorescent bead sample and generate a three-photon fluorescence signal.

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