CN108844929B - Method and device for detecting split pupil differential confocal split fluorescence spectrum and fluorescence life - Google Patents
Method and device for detecting split pupil differential confocal split fluorescence spectrum and fluorescence life Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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- G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
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Abstract
The invention belongs to the technical field of chemical substance detection, and utilizes a beam splitting pupil design to effectively shield the interference of autofluorescence of an optical element in an excitation light path on a result and improve the signal-to-noise ratio of a system. In addition, the differential confocal object surface positioning technology is fused with the discrete fluorescence spectrum and fluorescence lifetime measurement technology; the high-precision measurement of the three-dimensional appearance of the surface of the sample to be detected is realized by utilizing a differential confocal technology, and the high-sensitivity detection of the fluorescence spectrum and the fluorescence lifetime of each point on the surface of the sample to be detected is realized by utilizing a discrete fluorescence spectrum and fluorescence lifetime detection technology, so that the three-dimensional high-resolution spatial material composition distribution information is obtained. In the process of measuring the fluorescence information on the surface of the sample, the invention uses a plurality of different discrete fluorescence detection means, and a user can select whether to use fluorescence spectrum detection or fluorescence lifetime detection according to the chemical characteristics of the substance to be detected. The invention has wide application prospect in the fields of biology, medicine, material science and clinical medical diagnosis.
Description
Technical Field
The invention belongs to the technical field of chemical substance detection, can not only identify the components of substances with high sensitivity, but also detect the spatial distribution of the components of the substances, and has important application in the research fields of biology, medicine, material science and the like and the aspect of clinical medical diagnosis.
Background
In the fields of biology, physics, chemistry and materials, spectroscopic detection and analysis have become a fundamental measurement tool for basic research due to their very high sensitivity, molecular specificity and non-contact measurement characteristics. The fluorescence spectrum detection is a method for qualitatively and quantitatively analyzing substances by using the characteristics and the intensity of fluorescence generated by the substances under the irradiation of ultraviolet light. Especially for most organic compounds, the generated fluorescence usually has strong specificity and directivity. Therefore, the fluorescence spectrum detection is particularly suitable for the component analysis of the organic compounds with strong fluorescence specificity. In the industry, fluorescence spectroscopy can be used for component detection of various pollutants, component detection of industrial raw materials, component detection of petroleum crude oil, and the like. In the field of food safety, fluorescence spectrum detection can be used for detecting and monitoring information of mildew bacteria, pesticide residue and the like of food. In the medical field, fluorescence spectroscopy and fluorescence microscopy provide new directions for real-time imaging and detection of tumors and cancers, and are expected to be developed into medical technologies suitable for clinical diagnosis. Therefore, the fluorescence spectrum detection technology is a very practical and promising optical detection means.
Generally, fluorescence measurement techniques include both fluorescence spectroscopy measurements and fluorescence lifetime measurements. The fluorescence spectrum measurement technology is realized by detecting the spectral distribution of fluorescence emitted from a sample to be detected, namely, the excitation wavelength is fixed, and the relation curve of the emitted light intensity and the incident light wavelength of the sample is detected. The fluorescence spectrum technology is often combined with a fluorescence probe and applied to the fields of DNA sequencing, high polymer material science, biological fluorescence imaging and the like. In contrast, the fluorescence lifetime detection is a time required for the intensity of fluorescence excited from a sample to be examined to decrease to 1/e of the maximum intensity of fluorescence at the time of excitation after the excitation by a pulse light source. The fluorescence lifetime of the fluorescent substance is directly related to the structure of the fluorescent substance, the polarity of the microenvironment, the viscosity and other conditions, so that the lifetime value of fluorescence excited from the sample is absolute, is not influenced by factors such as excitation light intensity, fluorophore concentration and photobleaching, and is not limited by other factors for limiting intensity measurement. The fluorescence lifetime measurement of the sample can also be used for quantitatively measuring the distribution of a plurality of biochemical parameters such as pH value, ion concentration, temperature and the like of the microenvironment of the sample to be measured.
However, in the process of scanning and imaging the surface-excited fluorescence of the sample to be detected, when the surface of the sample to be detected has unevenness, it is not possible to ensure that the sizes of the light spots of the excitation light beam at different positions on the surface of the sample to be detected are consistent, and thus the resolution of the detection system at different positions cannot be kept consistent. Especially for some samples with large fluctuation, even the possibility that the objective lens collides with a sample to be measured in the sample scanning process due to the small working distance of the objective lens measured by the system exists, and finally, not only can the fluorescence distribution image on the surface of the sample not be obtained, but also the surface of the objective lens of the system is polluted. In addition, when a weak fluorescence signal is detected, on the excitation light path, the strong light can not only excite the fluorescence of the sample, but also excite the autofluorescence of the optical element, thereby interfering with the experimental result and reducing the signal-to-noise ratio of the system.
The design of the beam splitting pupil is beneficial to shielding the autofluorescence on an excitation light path, improving the signal-to-noise ratio of the system and realizing high-sensitivity detection. The confocal technology adopts a pinhole to filter scattered light outside a focus, has higher transverse resolution compared with the traditional microscope, and has unique axial chromatographic capacity. The differential confocal technology is that two pinholes which are symmetrical about an optical axis are added in a detection light path on the basis of the confocal technology, each pinhole corresponds to a light intensity sensor, and the distance of an object deviating from a focal plane is reflected by detecting the difference value of the light intensities of two paths penetrating through the pinhole, namely the differential confocal response value of a system. The linear range and axial resolution of the differential confocal response curve are twice those of the traditional confocal technology; the differential mode can effectively inhibit the drift of the light source and the electronic drift of the detector, and common-mode noise generated by the surface reflectivity fluctuation of the measured object, and the like, improves the signal-to-noise ratio and the stability of the system, and is widely applied to the field of non-contact measurement.
If the differential confocal technology with precise axial resolution and the design of the beam splitting pupil can be applied to the fluorescence scanning imaging system, the fluorescence imaging system can be ensured to ensure the same transverse resolution at each position of the surface of the sample to be detected, the possibility that an objective lens impacts the sample to be detected with larger surface fluctuation in the scanning process is effectively avoided, the autofluorescence of an optical element can be effectively shielded, and the signal-to-noise ratio of the system is improved. Meanwhile, after the whole scanning measurement process is finished, the high-resolution object three-dimensional shape information of the sample to be measured can be obtained at the same time, and the measured fluorescence spectrum distribution can be accurately corresponding to the three-dimensional shape. The method has great significance for comprehensively and accurately analyzing the spatial component distribution of the sample to be detected, and can be widely applied to the research fields of biology, materials science, medicine and the like.
Disclosure of Invention
The invention provides a method and a device for detecting a spectroscopic pupil differential confocal discrete fluorescence spectrum and a fluorescence life, which aim to solve the problems of measurement of a surface fluorescence spectrum and a fluorescence life of a sample to be detected in a three-dimensional space shape and interference of autofluorescence of an optical element in an excitation light path on a result and obtain three-dimensional shape information with high axial resolution of the sample to be detected and fluorescence spectrum information and fluorescence life information with high signal-to-noise ratio corresponding to each position.
The specific thought of the invention is as follows: by utilizing the design of the beam splitting pupil, the interference of the autofluorescence of the optical element in the excitation light path on the result is effectively shielded, and the signal-to-noise ratio of the system is improved. In addition, the differential confocal object surface positioning technology is fused with the discrete fluorescence spectrum and fluorescence lifetime measurement technology; the high-precision measurement of the three-dimensional appearance of the surface of the sample to be detected is realized by utilizing a differential confocal technology, and the high-sensitivity detection of the fluorescence spectrum and the fluorescence lifetime of each point on the surface of the sample to be detected is realized by utilizing a discrete fluorescence spectrum and fluorescence lifetime detection technology, so that the three-dimensional high-resolution spatial material composition distribution information is obtained.
On one hand, the invention provides a pupil differential confocal discrete fluorescence spectrum and fluorescence life detection device, which comprises a pulse laser light source, a continuous laser light source, a first spectroscope, a beam expander, an illumination pupil, an objective lens, a collection pupil, a first reflector, a first dichroic spectroscope, a pupil differential confocal detection system, a discrete fluorescence spectrum and fluorescence life detection system, a three-dimensional translation table, a signal collector and a computer, wherein the first spectroscope, the beam expander, the illumination pupil, the objective lens, the collection pupil, the first reflector, the first dichroic spectroscope, the pupil differential confocal;
wherein an illumination pupil and a collection pupil are placed in a pupil plane of the objective lens; the first spectroscope combines pulse laser emitted by a pulse laser light source and continuous laser emitted by a continuous laser light source to form a composite light beam; the beam expander, the illumination pupil and the objective lens are sequentially positioned in the exit direction of the synthesized light beam, the synthesized light beam is expanded by the beam expander, and after passing through the illumination pupil, the synthesized light beam is converged by the objective lens to form a detection light beam to irradiate on a sample to be detected; backward scattered light and fluorescence excited from a sample to be detected are collected through the objective lens, pass through a collecting pupil and are reflected by a first reflecting mirror; the light beam reflected by the first reflector is split by the first dichroic beam splitter, one path of the light beam is an intrinsic light beam with the same wavelength as that of the detection light beam, the intrinsic light beam enters a beam splitting pupil differential confocal detection system, and the other path of the light beam is a fluorescent light beam with the wavelength different from that of the detection light beam, and the fluorescent light beam enters a discrete fluorescence spectrum and fluorescence life detection system;
a sample to be detected is placed on the three-dimensional translation table, and the three-dimensional translation table is controlled by a computer to drive the sample to be detected to scan and move along three spatial directions; the signal collector converts a light intensity response signal which is obtained by the detection of the beam splitting pupil differential confocal detection system and changes along with the height of the sample to be detected, transmits the converted light intensity response signal to the computer, obtains a differential confocal response curve after the processing of the computer, and accurately determines the height value of each point on the surface of the sample to be detected through the zero point position of the differential confocal response curve; when the detection light beam is focused on each sampling point position on the surface of a sample to be detected, the signal collector converts fluorescence intensity information which is obtained by the discrete fluorescence spectrum and the fluorescence lifetime detection system and changes along with time under different wavelengths, and transmits the converted fluorescence intensity information to the computer, and the fluorescence lifetime and the fluorescence spectrum under different wavelengths, which are excited from the sampling points, are obtained after the conversion by the computer; and then the three-dimensional appearance of the surface of the sample, the fluorescence life of each point on the surface under different wavelengths and the fluorescence spectrum are obtained through scanning.
On the other hand, the invention also provides a pupil differential confocal discrete fluorescence spectrum and fluorescence lifetime detection method, which comprises the following operation steps:
(a) combining pulse laser emitted by a pulse laser light source and continuous laser emitted by a continuous laser light source through a first spectroscope to form a composite beam, wherein the pulse laser and the continuous laser have the same wavelength; the synthesized light beam is expanded by the beam expander, passes through the illumination pupil, and is converged by the objective lens to form a detection light beam to irradiate on a sample to be detected; defining two orthogonal directions perpendicular to the optical axis of the objective lens asxAndyin a direction along the optical axis of the objective lenszDirection;
(b) the backward scattering light generated by irradiating a sample with a light beam and the fluorescence excited by the sample to be detected are collected together through an objective lens, then pass through a collection pupil and are reflected by a first reflector, the light beam reflected by the first reflector is divided into two paths after passing through a first dichroic beam splitter, one path is an intrinsic light beam with the same wavelength as the detection light beam, and the other path is a fluorescence light beam with the wavelength different from the detection light beam; the intrinsic light beam enters a spectral pupil differential confocal detection system, and the fluorescent light beam enters a discrete fluorescence spectrum and fluorescence lifetime detection system;
(c) turn on the continuous laser light source alongxAndymoving the sample to be measured to the starting position of the transverse scanning (x 1,y 1) Then at the position edgezScanning a sample to be detected in a direction, measuring a differential confocal response curve which changes along with the scanning position by using a spectral pupil differential confocal detection system, and further accurately determining the surface position of the sample to be detected on which a detection light beam is focused according to the zero point position of the differential confocal response curve;
(d) closing the continuous laser light source, moving the sample to be detected according to the measurement result in the step (c), focusing the detection light beam on the surface of the sample to be detected, controlling the pulse laser light source to emit pulse laser, exciting fluorescence on the surface of the sample to be detected by the pulse laser, and obtaining fluorescence light intensity information which changes along with time under different wavelengths through a discrete fluorescence spectrum and fluorescence life detection system; and performing data analysis on the information to obtain the fluorescence life under different wavelengths;
(e) edge ofxAndyscanning the sample to be measured in the direction, repeating the above steps, and (b) at each scanning pointx i ,y i ) Determining the surface information of the sample to be detected at the position by using the zero position of the differential confocal response curve at the position, and measuring the fluorescence life of the fluorescence excited from the position under different wavelengths by using a discrete fluorescence spectrum and fluorescence life detection system;
(f) the obtained sample to be measured is arranged at each scanning point (x i ,y i ) And reconstructing the surface position information of the position and the corresponding fluorescence lifetime information, and simultaneously obtaining the three-dimensional appearance profile of the detected sample and the fluorescence lifetime of each point on the surface of the detected sample under different wavelengths.
Compared with the prior art, the invention has the following innovation points:
1. the differential confocal technology is combined with the fluorescence spectrum and service life detection technology, so that high-sensitivity detection of the material components can be realized, three-dimensional space distribution information of the material components can be detected, and the provided information is richer;
2. the interference of secondary fluorescence signals generated by an optical element on an excitation light path on an experimental result is effectively shielded by using a beam splitting pupil design, so that the signal-to-noise ratio of the system is greatly improved;
3. the service life of fluorescence under different wavelengths is detected by using a plurality of light intensity sensors at the same time, and the chemical components of the sample to be detected are identified based on the service life, so that the identification speed and the identification accuracy are higher;
4. the invention can detect the fluorescence life of the sample to be detected under different wavelengths and the relative fluorescence intensity information among different wavelengths, and has large information quantity.
Compared with the prior art, the invention has the following remarkable advantages:
1. the spatial appearance measurement of a three-dimensional sample to be measured and the fluorescence spectrum and fluorescence life measurement of each point on the spatial surface can be realized simultaneously, and richer information is provided for the three-dimensional chemical component analysis of the sample;
2. differential confocal detection has better signal-to-noise ratio and stability, and is beneficial to accurately positioning the spatial distribution of the substance;
3. the invention can be used for high-sensitivity measurement of autofluorescence, the chemical components of the sample to be detected are detected by using the autofluorescence of the sample to be detected, and a fluorescent marker is not needed in the detection process, so the detection process is very convenient.
Drawings
FIG. 1 is a schematic diagram of a pupil-splitting differential confocal split fluorescence spectroscopy and fluorescence lifetime detection method according to the present invention;
FIG. 2 is a schematic diagram of a pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection apparatus according to the present invention;
FIG. 3 is a schematic diagram of a spectroscopic pupil differential confocal detection system according to the present invention;
FIG. 4 is a diagram of a discrete fluorescence spectroscopy and fluorescence lifetime detection system of the present invention utilizing a dichroic beamsplitter, a narrowband filter, and a light intensity sensor;
FIG. 5 is a discrete fluorescence spectroscopy and fluorescence lifetime detection system of the present invention using a dichroic beamsplitter, a narrowband filter, a converging lens, a pinhole, and a light intensity sensor;
FIG. 6 is a diagram of a multi-intensity sensor set for use in a discrete fluorescence spectroscopy and fluorescence lifetime detection system of the present inventionNA schematic diagram of a light intensity sensor;
FIG. 7 is a diagram of a discrete fluorescence spectroscopy and fluorescence lifetime detection system of the present invention using a dichroic beamsplitter, a narrowband filter, a fiber focusing lens, a fiber delay line, and a light intensity sensor;
FIG. 8 is a diagram of a discrete fluorescence spectroscopy and fluorescence lifetime detection system of the present invention using a filter wheel and a light intensity sensor;
FIG. 9 is a schematic diagram of a pupil-splitting differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection embodiment of the present invention;
FIG. 10 shows a pupil-dividing differential confocal response curve FES (of the present invention)z) A schematic diagram of (a);
wherein: 1-pulse laser light source, 2-continuous laser light source, 3-first spectroscope, 4-beam expander, 5-lighting pupil, 6-collecting pupil, 7-objective, 8-sample to be measured, 9-three-dimensional translation stage, 10-first reflector, 11-first dichroic spectroscope, 12-spectral pupil differential confocal detection system, 13-discrete fluorescence spectrum and fluorescence lifetime detection system, 14-signal collector, 15-computer, 16-differential confocal converging lens, 17-double-hole pinhole, 18-first differential confocal light intensity sensor, 19-second differential confocal light intensity sensor, 20-second dichroic spectroscope, 21-third dichroic spectroscope, 22-NDichroic beam splitter, No. 23-narrow band filter, No. 24-narrow band filter, and No. 25-, (N-1) narrow band filter, 26-NNo. 27 narrow band filter, No. 27-first light intensity sensor, No. 28-second light intensity sensor, and No. 29-, (N-1) light intensity sensor, 30-NLuminous intensity sensor, 31-first convergent lens, 32-second convergent lens, 33-, (N-1) converging lens, 34-NConvergent lens, 35-pinhole, 36-pinhole, 37-, (NPinhole No. 1, 38-NPinhole, 39-second reflector, 40-multi-light intensity sensor group, 41-first fiber focusing lens, 42-second fiber focusing lens, 43-the (th) ((III))N-1) a fiber focusing lens, 44-thNOptical fiber focusingLens, 45-first fiber delay line, 46-second fiber delay line, 47-the (c)N-1) optical fiber delay line, 48-thNFiber delay line, 49-, (N+ 1) light intensity sensor, 50-filter wheel, 51-, (51-)N+ 2) light intensity sensor, No. 52-fourth dichroic beam splitter, No. 53-third narrow band filter, No. 54-fourth narrow band filter, 55-first photomultiplier, 56-second photomultiplier, 57-third photomultiplier, 58-fourth photomultiplier.
Detailed Description
The invention is further illustrated by the following figures and examples.
The basic idea of the invention is to effectively shield the autofluorescence of the optical element of the excitation light path by using the design of the pupil division, and improve the signal-to-noise ratio of the system; the method combines a confocal object surface positioning technology with precise axial resolution with a discrete fluorescence spectrum and fluorescence life measuring technology, utilizes a differential confocal technology to solve the high-precision measurement of the three-dimensional appearance of the surface of a sample to be measured, and simultaneously utilizes a discrete fluorescence spectrum and fluorescence life detecting technology to solve the high-sensitivity detection of the fluorescence spectrum and the fluorescence life of each point on the surface of the sample to be measured, thereby obtaining the three-dimensional high-resolution spatial material component distribution information. In the process of measuring the fluorescence information on the surface of the sample, the invention uses a plurality of different discrete fluorescence detection means, and a user can select the detection means according to specific application requirements. Meanwhile, the user can select whether to use fluorescence spectrum detection or fluorescence lifetime detection or the combination of the fluorescence spectrum detection and the fluorescence lifetime detection to identify the substance components according to the chemical characteristics of the substance to be detected.
Example 1
The problem to be solved in this embodiment is to scan the three-dimensional shape of the sample to be detected and analyze the spatial distribution of the tumor tissue in the sample to be detected, and accordingly determine the boundary information of the tumor tissue. In the embodiment, a pupil-splitting differential confocal detection system is used for measuring the three-dimensional morphology, and the fluorescence lifetime of four wavelengths, namely 400nm, 450nm, 530 nm and 580nm, excited by 355 nm wavelength pulse laser in a sample is used for judging whether each scanning point is a tumor cell. Since the fluorescence signal of the sample is very weak, the present embodiment uses the photomultiplier tube as the light intensity sensor for fluorescence detection to improve the fluorescence light intensity detection sensitivity of the system. Fig. 9 is a specific implementation device of this embodiment when realizing split-pupil differential confocal split fluorescence spectrum and fluorescence lifetime detection, including a pulse laser light source 1, a continuous laser light source 2, a first beam splitter 3, a beam expander 4, an illumination pupil 5, a collection pupil 6, an objective lens 7, a three-dimensional translation stage 9, a first reflector 10, a first dichroic beam splitter 11, a split-pupil differential confocal detection system 12, a split fluorescence spectrum and fluorescence lifetime detection system 13, a signal collector 14, and a computer 15. The wavelengths of the pulse laser light source 1 and the continuous laser light source 2 are both 355 nm, and the pulse width of the pulse laser light source 1 is 2 ns. Laser emitted by the pulse laser source 1 and the continuous laser source 2 is combined by the first beam splitter 3 and then sequentially passes through the beam expander 4, the illumination pupil 5 and the objective lens 7 to form a detection beam to irradiate on a sample 8 to be detected. The sample 8 to be measured is placed on the three-dimensional translation stage 9, and is scanned by the three-dimensional translation stage 9. The backward scattering light generated by the light beam irradiating the sample 8 to be detected and the fluorescence excited from the sample 8 to be detected are collected by the objective lens 7, reflected by the first reflecting mirror 10 after passing through the collection pupil 6, and then divided into two paths after passing through the first dichroic beam splitter 11, wherein one path is an intrinsic light beam with the same wavelength as the detection light beam and enters the beam splitting pupil differential confocal detection system 12, and the other path is a fluorescence light beam with the wavelength different from the detection light beam and enters the discrete fluorescence spectrum and fluorescence lifetime detection system 13.
In the system, the pupil differential confocal detection system 12 comprises a differential confocal converging lens 16, a double-hole pinhole 17, a first differential confocal light intensity sensor 18 and a second differential confocal light intensity sensor 19. Wherein two pinholes of the two-pinhole pinholes 17 are located on the focal plane of the differential confocal converging lens 16 and are placed in bilateral symmetry about the optical axis of the differential confocal converging lens 16. The first differential confocal light intensity sensor 18 and the second differential confocal light intensity sensor 19 are respectively displaced behind the holes of the double-hole pinhole. The differential confocal focusing lens 16 focuses the light beam entering the split-pupil differential confocal detection system 12 alongzThe sample being scanned axially, the beams passing through two-hole pinholes 17 respectivelyTwo pinholes are detected by a first differential confocal light intensity sensor 18 and a second differential confocal light intensity sensor 19 respectively.
The discrete fluorescence spectrum and fluorescence lifetime detection system 13 includes a dichroic beam splitter No. two 20, a narrow band filter No. one 23, a first photomultiplier 55, a dichroic beam splitter No. three 21, a narrow band filter No. two 24, a second photomultiplier 56, a dichroic beam splitter No. four 52, a narrow band filter No. three 53, a third photomultiplier 57, a narrow band filter No. four 54, and a fourth photomultiplier 58. The light beam entering the discrete fluorescence spectrum and fluorescence lifetime detection system 13 is split by 3 times by the second dichroic beam splitter 20, the third dichroic beam splitter 21 and the fourth dichroic beam splitter 52, so as to obtain 4 paths of fluorescence light beams with different wavelength bands. The 4 paths of fluorescent light beams with different wavelength bands are respectively detected and received by a first photomultiplier 55, a second photomultiplier 56, a third photomultiplier 57 and a fourth photomultiplier 58 after passing through a first narrowband filter 23, a second narrowband filter 24, a third narrowband filter 53 and a fourth narrowband filter 54. The center wavelengths corresponding to the narrow-band filters are 400nm, 450nm, 530 nm and 580nm respectively. The signal collector 14 is used for collecting the light intensity information collected by each light intensity sensor in the pupil-dividing differential confocal detection system 12 and the discrete fluorescence spectrum and fluorescence lifetime detection system 13, and transmitting the light intensity information to the computer 15 after converting the light intensity information. The computer 15 analyzes the acquired light intensity information to obtain a confocal response curve and fluorescence light intensity information which changes with time under each wavelength. Defining two orthogonal directions perpendicular to the optical axis of the objective lens 7 asxAndyin a direction along the optical axis of the objective lens 7zAnd (4) direction.
The method comprises the following steps of identifying the components of a sample to be detected.
(a) Turn on the continuous laser light source 2 alongxAndymoving the sample 8 to be measured to the horizontal scanning start position (x 1 , y 1) Then at the position edgezThe sample 8 to be measured is directionally scanned. Using the spectroscopic pupil differential confocal detection system 12, the differential confocal response curve FES (shown in FIG. 10) as a function of the scanning position is measuredz) And further according to a differential confocal response curve FES: (z) OfAccurately determining the position of the probe beam focused on the surface of the sample to be measured by the point response point, and recording the scanning position (x 1 , y 1) At a surface position of the sample 8 to be measured of a height ofz 1。
(b) And (b) closing the continuous laser light source, moving the sample 8 to be detected according to the measurement result in the step (a), focusing the detection light beam on the surface of the sample 8 to be detected, controlling the pulse laser light source 1 to emit pulse laser, exciting fluorescence on the surface of the sample 8 to be detected by the pulse laser, collecting the excited fluorescence by the objective lens 7, reflecting the fluorescence by the first reflecting mirror 10 through the collecting pupil 6, and then entering the discrete fluorescence spectrum and fluorescence lifetime detection system 13 through the first dichroic spectroscope 11. The fluorescence entering the discrete fluorescence spectrum and fluorescence lifetime detection system 13 is divided into two paths after passing through a second dichroic beam splitter 20, wherein the wavelength range of the reflected light beam is 360 nm-430 nm, and the wavelength range of the transmitted light beam is 430 nm-700 nm; the light beam transmitted by the second dichroic beam splitter 20 passes through the third dichroic beam splitter 21 and then is divided into two paths, wherein the wavelength range of the reflected light beam is 430 nm-480 nm, and the wavelength range of the transmitted light beam is 480 nm-700 nm; the light beam transmitted by the third dichroic beam splitter passes through the fourth dichroic beam splitter 54 and then is divided into two paths, wherein the wavelength range of the reflected light beam is 480 nm-550 nm, and the wavelength range of the transmitted light beam is 550 nm-700 nm. The light beam reflected by the second dichroic beam splitter 20 passes through the first narrowband filter 23 and then is irradiated onto the first photomultiplier 57. The center wavelength of the first narrowband filter 23 is 400nm, and the bandpass width is 10 nm. The central wavelength of the fluorescence received by the first photomultiplier tube 55 is therefore 400 nm. The light beam reflected by the third dichroic beam splitter 21 passes through the second narrowband filter 24 and then impinges on the second photomultiplier 56. The center wavelength of the second narrow band filter 24 is 450nm, and the band-pass width is 10 nm. The central wavelength of the fluorescence received by the second photomultiplier tube 56 is therefore 450 nm. The light beam reflected by the fourth dichroic beam splitter 52 passes through a third narrow band filter 53 and then irradiates a third photomultiplier 57; the center wavelength of the third narrowband filter 54 is 530 nm, and the bandpass width is 10 nm. The central wavelength of the fluorescence received by the third photomultiplier tube 57 is therefore 530 nm. The light beam transmitted by the fourth dichroic beam splitter 52 passes through a fourth narrowband filter 54 and then irradiates a fourth photomultiplier 58; the center wavelength of the fourth narrow band filter 54 is 580nm, and the band-pass width is 10 nm; the central wavelength of the fluorescence received by the fourth photomultiplier tube 58 is therefore 580 nm.
(c) The fluorescence information with the time change at the central wavelength of 400nm detected by the first photomultiplier tube 55, the fluorescence information with the time change at the central wavelength of 450nm detected by the second photomultiplier tube 56, the fluorescence information with the time change at the central wavelength of 530 nm detected by the third photomultiplier tube 57, and the fluorescence information with the time change at the central wavelength of 580nm detected by the fourth photomultiplier tube 58 are simultaneously collected by the signal collector 14 and transmitted to the computer 15. The computer 15 processes the fluorescence signals varying with time at these different center wavelengths to obtain the fluorescence lifetime corresponding to each wavelength. Wherein the fluorescence lifetime corresponding to the central wavelength of 400nm is 10.5 ns, the fluorescence lifetime corresponding to the central wavelength of 450nm is 7.3 ns, the fluorescence lifetime corresponding to the central wavelength of 530 nm is 13.3 ns, and the fluorescence lifetime corresponding to the central wavelength of 580nm is 6.7 ns.
(d) According to the fluorescence lifetime of normal tissue fluorescence at each wavelength: the fluorescence lifetime corresponding to the wavelength of 400nm is 9 ns-13 ns, the fluorescence lifetime corresponding to the wavelength of 450nm is 10 ns-14 ns, the fluorescence lifetime corresponding to the wavelength of 530 nm is 12 ns-15 ns, and the fluorescence lifetime corresponding to the wavelength of 580nm is 11 ns-14 ns; fluorescence lifetime of tumor tissue fluorescence at each wavelength: the fluorescence lifetime corresponding to the wavelength of 400nm is 8 ns-11 ns, the fluorescence lifetime corresponding to the wavelength of 450nm is 6 ns-8 ns, the fluorescence lifetime corresponding to the wavelength of 530 nm is 13 ns-16 ns, and the fluorescence lifetime corresponding to the wavelength of 580nm is 5 ns-7 ns; can obtain the surface (of the sample 7 to be measured)x ,1 y 1 , z 1) The fluorescence lifetime information of the tumor tissue is completely matched with the fluorescence lifetime information of the tumor tissue, so that the tumor tissue can be judged to be inx 1 , y 1 , z 1) Tumor tissue is treated.
(e) Edge ofxAndydirection scanning to be measuredSample, repeat the above steps, at each scanning point: (x i , y i ) Determining the surface information of the sample 8 to be measured at the position by using a spectroscopic pupil differential confocal detection system 12 at the positionz i The fluorescence lifetime of the fluorescence excited from the position at different wavelengths is measured by the discrete fluorescence spectroscopy and fluorescence lifetime detection system 13, and based on this, it is determined whether the position is normal tissue or tumor tissue.
(f) At each scanning point of the sample to be measured obtained by the information (b)x i , y i ) Surface position information of a locationz i And reconstructing the corresponding fluorescence lifetime information, obtaining the three-dimensional profile of the tested sample 8 and the fluorescence lifetime of each point on the surface of the tested sample under different wavelengths, and obtaining the judgment result of whether each point on the surface is a tumor tissue or a normal tissue. Further, based on the information, the spatial distribution of the tumor tissue in the sample 8 to be tested and the boundary information of the tumor tissue can be obtained.
Example 2
Unlike embodiment 1, this example uses a discrete fluorescence spectrum to determine whether each point on the surface of the sample 8 to be measured is tumor tissue or normal tissue. The apparatus and sample used were the same as in example 1. In order to improve the stability of the fluorescence spectrum measurement, the continuous laser light source 2 is used to emit a light beam to excite the sample 8 to be measured to generate fluorescence, and the measurement steps are as follows.
(a) Turn on the continuous laser light source 2 alongxAndymoving the sample 8 to be measured to the horizontal scanning start position (x 1 , y 1) Then at the position edgezThe sample 8 to be measured is directionally scanned. Using the spectroscopic pupil differential confocal detection system 12, the differential confocal response curve FES (shown in FIG. 10) as a function of the scanning position is measuredz) And further according to the differential confocal response curve FES: (z) Precisely determining the position of the probe beam focused on the surface of the sample to be measured, and recording the scanning position (x 1 , y 1) At a surface position of the sample 8 to be measured of a height ofz 1。
(b) And (b) moving the sample 8 to be detected according to the measurement result in the step (a), so that the detection light beam is focused on the surface of the sample 8 to be detected, the continuous laser emitted by the continuous laser light source 2 irradiates the surface of the sample 8 to be detected to excite fluorescence, the excited fluorescence is collected by the objective lens 7, passes through the collection pupil 6, is reflected by the first reflector 10, and then enters the discrete fluorescence spectrum and fluorescence lifetime detection system 13 through the first dichroic spectroscope 9. The fluorescence entering the discrete fluorescence spectrum and fluorescence lifetime detection system 13 is divided into two paths after passing through a second dichroic beam splitter 20, wherein the wavelength range of the reflected light beam is 360 nm-430 nm, and the wavelength range of the transmitted light beam is 430 nm-700 nm; the light beam transmitted by the second dichroic beam splitter 20 passes through the third dichroic beam splitter 21 and then is divided into two paths, wherein the wavelength range of the reflected light beam is 430 nm-480 nm, and the wavelength range of the transmitted light beam is 480 nm-700 nm; the light beam transmitted by the third dichroic beam splitter 23 passes through the fourth dichroic beam splitter 54 and then is divided into two paths, wherein the wavelength range of the reflected light beam is 480 nm-550 nm, and the wavelength range of the transmitted light beam is 550 nm-700 nm. The light beam reflected by the second dichroic beam splitter 20 passes through the first narrowband filter 23 and then is irradiated onto the first photomultiplier tube 55. The center wavelength of the first narrowband filter 23 is 400nm, and the bandpass width is 10 nm. The central wavelength of the fluorescence received by the first photomultiplier tube 55 is therefore 400 nm. The light beam reflected by the third dichroic beam splitter 21 passes through the second narrowband filter 24 and then impinges on the second photomultiplier 56. The center wavelength of the second narrow band filter 24 is 450nm, and the band-pass width is 10 nm. The central wavelength of the fluorescence received by the second photomultiplier tube 56 is therefore 450 nm. The light beam reflected by the fourth dichroic beam splitter 52 passes through a third narrow band filter 53 and then irradiates a third photomultiplier 57; the center wavelength of the third narrowband filter 53 is 530 nm, and the bandpass width is 10 nm. The central wavelength of the fluorescence received by the third photomultiplier tube 57 is therefore 530 nm. The light beam transmitted by the fourth dichroic beam splitter 52 passes through a fourth narrowband filter 54 and then irradiates a fourth photomultiplier 58; the center wavelength of the fourth narrow band filter 54 is 580nm, and the band-pass width is 10 nm; the central wavelength of the fluorescence received by the fourth photomultiplier tube 58 is therefore 580 nm. The computer 15 processes the fluorescence signals at the different central wavelengths to obtain fluorescence intensity information corresponding to each wavelength. Wherein the fluorescence intensity corresponding to the central wavelength of 400nm is 5.6 muW; the fluorescence intensity corresponding to the central wavelength of 450nm is 8.4 muW; the fluorescence intensity corresponding to the central wavelength of 530 nm is 4.5 muW; the fluorescence intensity corresponding to a center wavelength of 580nm was 9.8 μ W. Further, the relative fluorescence intensity spectra of the sample 8 to be detected under 400nm, 450nm, 530 nm and 580nm are 0.57:0.86:0.46: 1.
(c) According to the fact that the peak fluorescence spectrum of the normal tissue in the four-wavelength fluorescence information is 530 nm, and the peak fluorescence spectrum of the tumor tissue in the four-wavelength fluorescence information is 580nm, the surface point of the sample 8 to be detected is obtained (the point is the point where the sample 8 to be detected is located), (the point is the point where the sample is located, and the point is the point where the sample isx 1 ,y 1 ,z 1) Tumor tissue is treated.
(d) Edge ofxAndyscanning the sample to be measured in the direction, repeating the above steps, and (b) at each scanning pointx i ,y i ) Determining surface information of the sample 8 at a position by using a differential confocal detection system 12z i The fluorescence intensities of the fluorescence excited from the position at different wavelengths are measured by the discrete fluorescence spectrum and fluorescence lifetime detection system 13, and based on this, whether the tissue is normal tissue or tumor tissue is determined.
(e) At each scanning point of the sample to be measured obtained by the information (b)x i ,y i ) Surface position information of a locationz i And reconstructing the corresponding fluorescence lifetime information, obtaining the three-dimensional appearance profile of the tested sample 8 and the discrete fluorescence spectrum of each point on the surface of the tested sample, and obtaining the judgment result of whether each point on the surface is a tumor tissue or a normal tissue. Further, based on the information, the spatial distribution of the tumor tissue in the sample 8 to be tested and the boundary information of the tumor tissue can be obtained.
Example 3
Unlike embodiment 1, as shown in fig. 5, in order to improve the resolution of the probe beam, a condenser lens and a pinhole are respectively added in front of all the intensity sensors. The pinhole is arranged at the focus position of the converging lens, and the converging lens converges the fluorescent light beams with different wavelengths and then carries out spatial filtering through the pinhole. Therefore, the fluorescence signals detected by the light intensity sensors are the filtered fluorescence intensity information, the filtered fluorescence accurately corresponds to the fluorescence signals excited by the focus of the detection light beam, and the fluorescence signals outside the focus are effectively shielded.
Example 4
Unlike embodiment 1, as shown in fig. 6, in order to simplify the system structure and reduce the system cost, in this embodiment, the array photomultiplier is used as a multi-light intensity sensor group to replace the four photomultiplier detectors, and different detection units in the array photomultiplier are used to respectively detect and obtain fluorescence information with different wavelengths.
Example 5
Unlike example 1, the discrete fluorescence spectroscopy and fluorescence lifetime detection system includes 3 dichroic beam splitters (in this example, 3 dichroic beam splitters, as shown in FIG. 7N= 4), 4 narrowband filters, 4 fiber focusing lenses, 4 fiber delay lines with different delay times, and oneN+ 1) light intensity sensor 49. The 3 dichroic beam splitters split the fluorescent light beams excited by the sample to be detected for 3 times to obtain 4 paths of fluorescent light beams with different wavelength bands; the 4 paths of fluorescent light beams with different wavelength bands are respectively filtered by 4 narrow-band filters and then coupled into 4 optical fiber delay lines with different delay times by 4 optical fiber focusing lenses. The 4 paths of fluorescence after being delayed by the optical fiber delay line are synthesized and output at the tail end of the optical fiber delay line, and the output is composed ofN+ 1) light intensity sensor 51 detects reception. At this time, fluorescence of different wavelengths will arrive at different times: (N+ 1) light intensity sensor 51, so that the fluorescence information at different wavelengths can be separated according to different time periods. The mode is beneficial to simplifying the system structure, reducing the system volume and reducing the system cost.
Example 6
Unlike embodiment 1, as shown in FIG. 8, a filter wheel 50 and (C)N+ 2) light intensity sensingThe device 51 constitutes a discrete fluorescence spectroscopy and fluorescence lifetime detection system 13. The filter wheel 50 consists of 4 narrow band filters with different center wavelengths (in this embodimentN= 4), each time the filter wheel 50 rotates, the pulse laser source emits a pulse laser, the fluorescent beam excited from the sample 8 to be measured penetrates the narrow-band filter under the corresponding central wavelength, (b) and (d)N+ 2) the light intensity sensor 51 measures the fluorescence light intensity information with time at the corresponding wavelength. Therefore, the filter wheel 50 can obtain the time-varying fluorescence intensity information at 4 different wavelengths after rotating for 4 times. By rotating the filter wheel 50, the number of light intensity sensors used can be reduced, thereby significantly reducing the system development cost.
Claims (10)
1. A device for detecting a pupil differential confocal discrete fluorescence spectrum and a fluorescence life comprises a pulse laser light source and a continuous laser light source, and is characterized in that: the system comprises a first beam splitter, a beam expander, an illumination pupil, an objective lens, a collection pupil, a first reflector, a first dichroic beam splitter, a beam splitting pupil differential confocal detection system, a discrete fluorescence spectrum and fluorescence life detection system, a three-dimensional translation stage, a signal collector and a computer;
wherein an illumination pupil and a collection pupil are placed in a pupil plane of the objective lens; the first spectroscope combines pulse laser emitted by a pulse laser light source and continuous laser emitted by a continuous laser light source to form a composite light beam; the beam expander, the illumination pupil and the objective lens are sequentially positioned in the exit direction of the synthesized light beam, the synthesized light beam is expanded by the beam expander, and after passing through the illumination pupil, the synthesized light beam is converged by the objective lens to form a detection light beam to irradiate on a sample to be detected; backward scattered light and fluorescence excited from a sample to be detected are collected through the objective lens, pass through a collecting pupil and are reflected by a first reflecting mirror; the light beam reflected by the first reflector is split by the first dichroic beam splitter, one path of the light beam is an intrinsic light beam with the same wavelength as that of the detection light beam, the intrinsic light beam enters a beam splitting pupil differential confocal detection system, and the other path of the light beam is a fluorescent light beam with the wavelength different from that of the detection light beam, and the fluorescent light beam enters a discrete fluorescence spectrum and fluorescence life detection system;
a sample to be detected is placed on the three-dimensional translation table, and the three-dimensional translation table is controlled by a computer to drive the sample to be detected to scan and move along three spatial directions; the signal collector converts a light intensity response signal which is obtained by the detection of the beam splitting pupil differential confocal detection system and changes along with the height of the sample to be detected, transmits the converted light intensity response signal to the computer, obtains a differential confocal response curve after the processing of the computer, and accurately determines the height value of each point on the surface of the sample to be detected through the zero point position of the differential confocal response curve; when the detection light beam is focused on each sampling point position on the surface of a sample to be detected, the signal collector converts fluorescence intensity information which is obtained by the discrete fluorescence spectrum and the fluorescence lifetime detection system and changes along with time under different wavelengths, and transmits the converted fluorescence intensity information to the computer, and the fluorescence lifetime and the fluorescence spectrum under different wavelengths, which are excited from the sampling points, are obtained after the conversion by the computer; and then the three-dimensional appearance of the surface of the sample, the fluorescence life of each point on the surface under different wavelengths and the fluorescence spectrum are obtained through scanning.
2. The split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection device of claim 1, wherein: the differential confocal detection system of the beam splitting pupil comprises a differential confocal convergent lens, a double-hole pinhole, a first differential confocal light intensity sensor and a second differential confocal light intensity sensor; two pinholes of the two-hole pinholes are positioned on a focal plane of the differential confocal converging lens and are symmetrically arranged on the left and right sides of the optical axis of the differential confocal converging lens; the first differential confocal light intensity sensor and the second differential confocal light intensity sensor are respectively positioned behind two pinholes of the double-hole pinhole; and subtracting the light intensity signal detected by the first differential confocal light intensity sensor from the light intensity signal obtained by the second differential confocal light intensity sensor to obtain a differential confocal response curve.
3. The split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection apparatus of claim 1, the discrete fluorescence spectroscopy and fluorescence lifetime detection system comprising (a)N-1) dichroic beam splitters,NA narrow band filter,NA light intensity sensor: from the following (N-1) a dichroic beam splitter for directing a fluorescent light beam emitted from a sample to be measuredN-1) sub-spectroscopy to obtainNFluorescent light beams with different wavelength bands; the above-mentionedNThe fluorescent light beams with different wavelength bands respectively pass throughNAfter being filtered by a narrow-band filterNThe light intensity sensor detects and receives to obtainNFluorescence intensity information varying with time at different wavelengths; each narrow band filter has a center wavelength ofλ n Band pass width ofλ n Wherein, in the step (A),n=1, 2, …,N。
4. the split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection device of claim 3, wherein: replacement of said use by a multi-intensity sensor groupNThe light intensity sensor: multiple light intensity sensor group consisting ofNAnd each light intensity sensor in the multi-light intensity sensor group is used for respectively detecting to obtain corresponding fluorescence intensity information.
5. The split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection device of claim 1, wherein: the discrete fluorescence spectroscopy and fluorescence lifetime detection system comprisesN-1) dichroic beam splitters,NA narrow band filter,NA fiber focusing lens,NA fiber delay line having different delay times andN+ 1) light intensity sensor: from the following (N-1) a dichroic beam splitter for directing a fluorescent light beam emitted from a sample to be measuredN-1) sub-spectroscopy to obtainNFluorescent light beams with different wavelength bands; the above-mentionedNThe fluorescent light beams with different wavelength bands respectively pass throughNAfter being filtered by a narrow-band filterNA fiber focusing lens coupled inNThe optical fiber delay lines have different delay times; after delay by optical fibre delay linesNThe path fluorescence is synthesized and output at the end of the optical fiber delay line, and is composed ofN+ 1) light intensity sensor detects and receives; at this time, fluorescence of different wavelengths will arrive at different times: (N+ 1) light intensity sensor, and therefore can be varied according to different conditionsSeparates the fluorescence information at different wavelengths.
6. The split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection device of claim 1, wherein: the discrete fluorescence spectrum and fluorescence lifetime detection system comprises a filter wheel and (A)N+ 2) light intensity sensor: the filter wheel is provided with a filter wheelNA narrow-band filter with different central wavelengths, wherein when the filter wheel rotates, the pulse laser source emits a pulse laser, the fluorescent beam excited from the sample to be measured passes through the narrow-band filter with the corresponding central wavelength (b)N+ 2) the light intensity sensor measures the fluorescence light intensity information which changes with time under the corresponding wavelength; rotation of the filter wheelNCan be obtained after a timeNFluorescence intensity information over time at different wavelengths.
7. The split-pupil differential confocal discrete fluorescence spectroscopy and fluorescence lifetime detection apparatus according to claim 3 or 6, wherein: a convergent lens and a pinhole are respectively added in front of each light intensity sensor: the pinhole is arranged at the focus position of the convergent lens, the convergent lens converges the fluorescent light beams with different wavelengths and then carries out spatial filtering through the pinhole, and the light intensity sensors detect the filtered fluorescent light intensity information.
8. The method for detecting the differential confocal discrete fluorescence spectrum of the pupil and the fluorescence life is characterized in that:
(a) combining pulse laser emitted by a pulse laser light source and continuous laser emitted by a continuous laser light source through a first spectroscope to form a composite beam, wherein the pulse laser and the continuous laser have the same wavelength; the synthesized light beam is expanded by the beam expander, passes through the illumination pupil, and is converged by the objective lens to form a detection light beam to irradiate on a sample to be detected; defining two orthogonal directions perpendicular to the optical axis of the objective lens asxAndyin a direction along the optical axis of the objective lenszDirection;
(b) the backward scattering light generated by irradiating a sample with a light beam and the fluorescence excited by the sample to be detected are collected together through an objective lens, then pass through a collection pupil and are reflected by a first reflector, the light beam reflected by the first reflector is divided into two paths after passing through a first dichroic beam splitter, one path is an intrinsic light beam with the same wavelength as the detection light beam, and the other path is a fluorescence light beam with the wavelength different from the detection light beam; the intrinsic light beam enters a spectral pupil differential confocal detection system, and the fluorescent light beam enters a discrete fluorescence spectrum and fluorescence lifetime detection system;
(c) turn on the continuous laser light source alongxAndymoving the sample to be measured to the starting position of the transverse scanning (x 1,y 1) Then at the position edgezScanning a sample to be detected in a direction, measuring a differential confocal response curve which changes along with the scanning position by using a spectral pupil differential confocal detection system, and further accurately determining the surface position of the sample to be detected on which a detection light beam is focused according to the zero point position of the differential confocal response curve;
(d) closing the continuous laser light source, moving the sample to be detected according to the measurement result in the step (c), focusing the detection light beam on the surface of the sample to be detected, controlling the pulse laser light source to emit pulse laser, exciting fluorescence on the surface of the sample to be detected by the pulse laser, and obtaining fluorescence light intensity information which changes along with time under different wavelengths through a discrete fluorescence spectrum and fluorescence life detection system; and performing data analysis on the information to obtain the fluorescence life under different wavelengths;
(e) edge ofxAndyscanning the sample to be measured in the direction, repeating the above steps, and (b) at each scanning pointx i ,y i ) Determining the surface information of the sample to be detected at the position by using the zero position of the differential confocal response curve at the position, and measuring the fluorescence life of the fluorescence excited from the position under different wavelengths by using a discrete fluorescence spectrum and fluorescence life detection system;
(f) the obtained sample to be measured is arranged at each scanning point (x i ,y i ) Reconstructing the surface position information of the position and the corresponding fluorescence life information, and simultaneously obtaining the three-dimensional appearance profile of the tested sample and the surface points of the tested sampleFluorescence lifetime at the same wavelength.
9. The method of claim 8, wherein the method comprises the steps of: at the zero point position of a differential confocal response curve of the pupil differential confocal system, the light spot of the detection light beam is accurately focused on the surface of a detection sample, the size of the focused light spot is minimum, and the detection area is minimum; the other positions of the differential confocal response curve correspond to the positions where the detection beams are focused on the deviated surface, and the positive and negative of the differential confocal response curve can reflect the direction of the objective lens deviated or close to the measured object; when fluorescence lifetime and relative fluorescence intensity spectrum information under different wavelengths at a certain surface position are measured, the size of a surface light spot of a detection light beam on a sample to be measured is controlled according to a differential confocal response curve, and then the size of a focused light spot is controlled according to actual measurement requirements, so that the size of a sample detection area is controlled.
10. The method for detecting the differential confocal discrete fluorescence spectrum of the pupil and the fluorescence life is characterized in that:
(a) combining pulse laser emitted by a pulse laser light source and continuous laser emitted by a continuous laser light source through a first spectroscope to form a composite beam, wherein the pulse laser and the continuous laser have the same wavelength; the synthesized light beam is expanded by the beam expander, passes through the illumination pupil, and is converged by the objective lens to form a detection light beam to irradiate on a sample to be detected; defining two orthogonal directions perpendicular to the optical axis of the objective lens asxAndyin a direction along the optical axis of the objective lenszDirection;
(b) the backward scattering light generated by irradiating the sample with the light beam and the fluorescence excited by the sample to be measured are collected together by the objective lens, pass through the collection pupil and are reflected by the first reflecting mirror; the light beam reflected by the first reflector is divided into two paths after passing through the first dichroic beam splitter, wherein one path is an intrinsic light beam with the same wavelength as the detection light beam, and the other path is a fluorescent light beam with the wavelength different from the detection light beam; the intrinsic light beam enters a spectral pupil differential confocal detection system, and the fluorescent light beam enters a discrete fluorescence spectrum and fluorescence lifetime detection system;
(c) turn on the continuous laser light source alongxAndymoving the sample to be measured to the starting position of the transverse scanning (x 1,y 1) Then at the position edgezScanning a sample to be detected in a direction, measuring a differential confocal response curve which changes along with the scanning position by using a spectral pupil differential confocal detection system, and further accurately determining the surface position of the sample to be detected on which a detection light beam is focused according to the zero point position of the differential confocal response curve;
(d) moving the sample to be measured according to the measurement result in the step (c), so that the detection light beam is focused on the surface of the sample to be measured, exciting fluorescence on the surface of the sample to be measured by continuous laser, obtaining fluorescence intensity information corresponding to different wavelengths through a discrete fluorescence spectrum and fluorescence life detection system, and analyzing the fluorescence intensity information to obtain discrete fluorescence spectrum information;
(e) edge ofxAndyscanning the sample to be measured in the direction, repeating the above steps, and (b) at each scanning pointx i, y i ) Determining the surface information of the sample to be detected at the position by using a confocal detection system, and measuring the discrete fluorescence spectrum information excited from the position by using a discrete fluorescence spectrum and fluorescence lifetime detection system;
(f) the obtained sample to be measured is arranged at each scanning point (x i ,y i ) And reconstructing the surface position information of the position and the corresponding discrete fluorescence spectrum information, and simultaneously obtaining the three-dimensional appearance profile of the detected sample and the discrete fluorescence spectrum of each point on the surface of the detected sample.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103439254A (en) * | 2013-09-06 | 2013-12-11 | 北京理工大学 | Spectroscopic pupil laser confocal Raman spectrum testing method and device |
| CN103884704A (en) * | 2014-03-10 | 2014-06-25 | 北京理工大学 | Spectral pupil laser confocal Brillouin-Raman spectrum measuring method and device |
| CN103969239A (en) * | 2013-09-06 | 2014-08-06 | 北京理工大学 | Split pupil laser differential confocal Raman spectrum test method and device |
| CN104482880A (en) * | 2014-12-17 | 2015-04-01 | 北京理工大学 | Laser stimulated emission depletion (STED) and three-dimensional superresolving spectral pupil differential confocal imaging method and device |
| CN104614353A (en) * | 2015-01-28 | 2015-05-13 | 中国科学院半导体研究所 | Two channel-based multi-spectrum fluorescent imaging microscopic system and method |
| CN105143855A (en) * | 2013-03-15 | 2015-12-09 | 雪松-西奈医学中心 | Time-resolved laser-induced fluorescence spectroscopy systems and uses thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN204731160U (en) * | 2015-06-01 | 2015-10-28 | 复旦大学 | A kind of autofluorescence life-span imaging and fluorescence spectrum combine the device being used for early diagnosis of cancer |
| CN107192702B (en) * | 2017-05-23 | 2020-02-04 | 北京理工大学 | Spectroscopic pupil laser confocal CARS (coherent anti-Raman scattering) microspectroscopy testing method and device |
-
2018
- 2018-05-14 CN CN201810452920.5A patent/CN108844929B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105143855A (en) * | 2013-03-15 | 2015-12-09 | 雪松-西奈医学中心 | Time-resolved laser-induced fluorescence spectroscopy systems and uses thereof |
| CN103439254A (en) * | 2013-09-06 | 2013-12-11 | 北京理工大学 | Spectroscopic pupil laser confocal Raman spectrum testing method and device |
| CN103969239A (en) * | 2013-09-06 | 2014-08-06 | 北京理工大学 | Split pupil laser differential confocal Raman spectrum test method and device |
| CN103884704A (en) * | 2014-03-10 | 2014-06-25 | 北京理工大学 | Spectral pupil laser confocal Brillouin-Raman spectrum measuring method and device |
| CN104482880A (en) * | 2014-12-17 | 2015-04-01 | 北京理工大学 | Laser stimulated emission depletion (STED) and three-dimensional superresolving spectral pupil differential confocal imaging method and device |
| CN104614353A (en) * | 2015-01-28 | 2015-05-13 | 中国科学院半导体研究所 | Two channel-based multi-spectrum fluorescent imaging microscopic system and method |
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