CN113484293B - Microscopic circular polarization fluorescence spectrum detection system and method based on single photon counting method - Google Patents
Microscopic circular polarization fluorescence spectrum detection system and method based on single photon counting method Download PDFInfo
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- CN113484293B CN113484293B CN202110841800.6A CN202110841800A CN113484293B CN 113484293 B CN113484293 B CN 113484293B CN 202110841800 A CN202110841800 A CN 202110841800A CN 113484293 B CN113484293 B CN 113484293B
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- 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
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Abstract
The invention relates to a microscopic circular polarization fluorescence spectrum detection system and a microscopic circular polarization fluorescence spectrum detection method based on a single photon counting method, wherein a white light source, a sample to be detected, a dichroic mirror, a photoelastic modulator and a gram prism are arranged in a line, light emitted by a switchable light source system is emitted into the dichroic mirror through a depolarizer, a detection switching mirror is movably arranged between the dichroic mirror and the photoelastic modulator, when the detection switching mirror is moved in, light emitted by the dichroic mirror is reflected into a microscopic imaging system through the detection switching mirror, when the detection switching mirror is moved out, light emitted by the dichroic mirror is emitted into a spectrum acquisition system through the photoelastic modulator and the gram prism, the photoelastic modulator is connected with the photoelastic modulator controller through a line, and the photoelastic modulator controller is connected with the spectrum acquisition system through a line. The invention can realize light source and system conversion as required, and can realize circular polarization fluorescence spectrum detection of solid samples, especially high-voltage module samples.
Description
Technical Field
The invention relates to the field of optical detection of samples, in particular to a microscopic circular polarization fluorescence spectrum detection system and method based on a single photon counting method.
Background
The circular polarization fluorescence spectrum detection system is a process of collecting the intensity of circular polarization fluorescence radiated by the depolarized excitation light after exciting a molecular characteristic spectrum of a sample, and can assist in analyzing the luminous characteristics of the material by detecting the circular polarization fluorescence spectrum.
The detection method of the circular polarization fluorescence spectrum comprises a lock-in amplification method, a gate integration method, a single photon counting method and the like, and in the methods, the single photon counting method has the advantages of high efficiency, adjustable integration area and the like.
Under the premise of adopting a single photon counting method, when detecting the circular polarized fluorescence spectrum, a xenon lamp or an LED light source is usually adopted to be matched with a monochromator for realizing, and the method can only be applied to circular polarized fluorescence spectrum detection of a liquid sample contained in a cuvette, cannot be applied to a solid sample, and cannot be used for microscopic in-situ detection of a target area.
However, for solid samples, since the distribution of the solid samples cannot be uniformly the same as that of the liquid samples, in-situ measurement is desired in repeated test, that is, excitation light in multiple measurement processes can strike the same positions of the samples, which requires excellent monochromaticity and coherence of the excitation light source, smaller focus can be obtained in focusing, and microscopic imaging can be performed on the sample area in the measurement process, but in the prior art, there is no detection system capable of simultaneously meeting the above requirements.
Disclosure of Invention
The invention aims to provide a microscopic circular polarization fluorescence spectrum detection system and method based on a single photon counting method, which can realize the conversion of a continuous laser light source and a detection system according to the requirement, wherein a microscopic imaging system is utilized to realize microscopic imaging and focusing of a sample area before measurement, and circular polarization fluorescence spectrum detection of a solid sample, particularly a high-voltage module sample can be realized.
The aim of the invention is realized by the following technical scheme:
The utility model provides a microscopic circular polarization fluorescence spectrum detecting system based on single photon counting method, includes switchable light source system, depolarizer, dichroscope, white light source, detects switching mirror, photoelastic modulator, gram prism, spectrum acquisition system and microscopic imaging system, wherein white light source, measured sample, dichroscope, photoelastic modulator and gram prism are the setting in a line, and switchable light source system locates dichroscope one side and the excitation light that jets out is penetrated into the dichroscope through the depolarizer, detects switching mirror movably locates between dichroscope and the photoelastic modulator, and when detecting switching mirror moves in, the exit light of dichroscope is through detecting switching mirror reflection is penetrated into microscopic imaging system, when detecting switching mirror moves out, the exit light of dichroscope passes through photoelastic modulator and gram prism are penetrated into in the spectrum acquisition system, photoelastic modulator passes through the circuit and photoelastic modulator controller links to each other, photoelastic modulator controller passes through the circuit and is connected with spectrum acquisition system.
The switchable light source system comprises a first continuous laser source, a second continuous laser source, a first reflecting mirror, a second reflecting mirror, a third reflecting mirror and a light source switching reflecting mirror, wherein the first reflecting mirror is arranged on an output light path of the first continuous laser source, the light source switching reflecting mirror is arranged on an output light path of the second continuous laser source and is movably arranged between the first reflecting mirror and the second reflecting mirror, when the light source switching reflecting mirror moves out, the emergent light of the first continuous laser source is reflected and injected into the depolarizer through the first reflecting mirror, the second reflecting mirror and the third reflecting mirror in sequence, and when the light source switching reflecting mirror moves in, the emergent light of the second continuous laser source is reflected and injected into the depolarizer through the light source switching reflecting mirror, the second reflecting mirror and the third reflecting mirror in sequence.
The microscopic imaging system comprises a fourth reflecting mirror, a second focusing lens, a CCD camera and a second computer, wherein light reflected by the detection switching reflecting mirror is reflected again by the fourth reflecting mirror and then focused by the second focusing lens to be emitted into the CCD camera, and the CCD camera is connected with the second computer through a circuit.
The spectrum acquisition system comprises a high-pass filter, a first focusing mirror, a light transmission optical fiber, a concave mirror, a light splitting grating, a photomultiplier, an acquisition board card and a first computer, wherein light beams screened by the gram prism are transmitted into the light transmission optical fiber through the high-pass filter and the first focusing mirror, are transmitted to the concave mirror through the light transmission optical fiber, are reflected to the light splitting grating through the concave mirror, are split into the photomultiplier through the light splitting grating, the photomultiplier is connected with the acquisition board card through a circuit, and the acquisition board card is connected with the first computer through the circuit.
The photoelastic modulator controller is connected with the acquisition board card through a circuit.
According to the method of the microscopic circular polarization fluorescence spectrum detection system based on the single photon counting method, the photoelastic modulator sends out periodic signals through a photoelastic modulator controller to control the periodic rotation, and the linear polarization fluorescence beam formed after passing through the photoelastic modulator and the gram prism obtains 2 times of strongest beam light intensity in one period, wherein one is the linear polarization fluorescence beam formed by the left-hand circular polarization fluorescence beam, the other is the linear polarization fluorescence beam formed by the right-hand circular polarization fluorescence beam, and the spectrum acquisition system acquires the linear polarization fluorescence beam and calculates to obtain the difference value between the relative light intensity (I L) of the left-hand circular polarization fluorescence beam spectrum and the relative light intensity (I R) of the right-hand circular polarization fluorescence beam spectrum, namely:
Δ＝IL-IR；
meanwhile, a fluorescence spectrum asymmetric system of the tested sample (10) is obtained, namely:
and obtaining the circular polarized fluorescence spectrum characteristic of the tested sample through analysis of the asymmetry coefficient.
The photoelastic modulator controller sends out a periodic square wave signal, and the photoelastic modulator is triggered by the rising edge of the square wave signal.
One signal period of the square wave signal is one rotation period of the optical axis of the photoelastic modulator, and the rotation period is 180 °.
The circular polarized fluorescent light beams corresponding to the optical axes of the photoelastic modulator and the Gray prism are left-handed circular polarized fluorescent light beams when the included angle between the optical axes of the photoelastic modulator and the Gray prism is 45 degrees, and the circular polarized fluorescent light beams corresponding to the optical axes of the photoelastic modulator and the Gray prism are right-handed circular polarized fluorescent light beams when the included angle between the optical axes of the photoelastic modulator and the Gray prism is 135 degrees.
The invention has the advantages and positive effects that:
1. The switchable light source system is provided with the light source switching reflector, and when the light source switching reflector moves in or out between the first reflector and the second reflector, the switching of different continuous laser light sources can be realized, so that the switching of multiple excitation wavelengths is realized.
2. The invention is provided with the detection switching reflector, when the detection switching reflector moves between the dichroic mirror and the photoelastic modulator, the white light source illuminates the sample to be detected, the sample to be detected is reflected by the detection switching reflector and then is incident into the microscopic imaging system for imaging, the selective focusing can be carried out, and when the detection switching reflector moves between the dichroic mirror and the photoelastic modulator, the excitation light beam is led into the sample to be detected, and the circular polarization fluorescence spectrum measurement of the sample to be detected can be carried out.
3. The related operation of the invention not only ensures the accurate incidence of the excitation light, but also compensates the technical blank that the circular polarized fluorescence spectrum of the solid sample cannot be measured, the in-situ measurement cannot be carried out in repeated experiments and the like in the prior art by matching the microscopic imaging with the continuous laser excitation light source.
Drawings
Figure 1 is a schematic view of the structure of the present invention,
Figure 2 is a schematic diagram of the working principle of the photoelastic modulator of figure 1,
Figure 3 is a schematic view of the fluorescence spectrum acquisition assembly of figure 1,
FIG. 4 is a schematic view of a circular polarized fluorescence spectrum of a sample to be tested obtained by the present invention.
The device comprises a first continuous laser source 1, a second continuous laser source 2, a first reflector 3, a light source switching reflector 4, a second reflector 5, a third reflector 6, a depolarizer 7, a dichroic mirror 8, an objective lens 9, a measured sample 10, a white light source 11, a detection switching reflector 12, a photoelastic modulator 13, a gram prism 14, a high-pass filter 15, a first focusing mirror 16, an input end 17, a light transmitting optical fiber 18, a concave mirror 19, a spectrograting 20, a photomultiplier 21, a first data transmission line 22, an acquisition board card 23, a second data transmission line 24, a first computer 25, a third data transmission line 26, a photoelastic modulator controller 27, a fourth data transmission line 28, a fourth reflector 29, a second focusing mirror 30, a CCD camera 31, a fifth data transmission line 32 and a second computer 33.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings.
As shown in fig. 1 to 4, the invention comprises a switchable light source system, a depolarizer 7, a dichroic mirror 8, a white light source 11, a detection switching reflector 12, a photoelastic modulator 13, a graticule prism 14, a spectrum acquisition system and a microscopic imaging system, wherein the white light source 11, a measured sample 10, the dichroic mirror 8, the detection switching reflector 12, the photoelastic modulator 13 and the graticule prism 14 are arranged in a line, the emergent light of the white light source 11 passes through the measured sample 10 and then enters the dichroic mirror 8, the switchable light source system is arranged at one side of the dichroic mirror 8 and can be switched to output excitation light sources with different wavelengths, the excitation light emitted by the switchable light source system is injected into the dichroic mirror 8 through the depolarizer 7, the detection switching reflector 12 is movably arranged between the dichroic mirror 8 and the photoelastic modulator 13, when the detection switching reflector 12 moves between the dichroic mirror 8 and the photoelastic modulator 13, the emergent light of the dichroic mirror 8 is reflected into the microscopic imaging system through the detection switching reflector 12, when the detection switching reflector 12 moves out of the dichroic mirror 8 and the photoelastic modulator 13, the emergent light of the dichroic mirror 8 passes through the dichroic mirror 8 and the photoelastic modulator 13 passes through the grating modulator 27, and the light modulator 27 is sequentially connected with the spectrum acquisition system, and the light modulator is connected with the light modulator 28 in turn, and the light modulator is connected with the light modulator 27 and the light modulator is connected with the light modulator 28 in sequence, and the light modulator 13 is connected with the light modulator system.
As shown in fig. 1, the switchable light source system includes a first continuous laser light source 1, a second continuous laser light source 2, a first mirror 3, a second mirror 5, a third mirror 6, and a light source switching mirror 4, wherein the first mirror 3 is disposed on an output light path of the first continuous laser light source 1, the light source switching mirror 4 is disposed on an output light path of the second continuous laser light source 2 and is movably disposed between the first mirror 3 and the second mirror 5, when the light source switching mirror 4 moves out between the first mirror 3 and the second mirror 5, the outgoing light of the first continuous laser light source 1 is reflected into the depolarizer 7 sequentially through the first mirror 3, the second mirror 5, and the third mirror 6, and when the light source switching mirror 4 moves into between the first mirror 3 and the second mirror 5, the outgoing light of the second continuous laser light source 2 sequentially passes through the light source switching mirror 4, the second mirror 5, and the third mirror 6 and is reflected into the depolarizer 7, thereby realizing the excitation of different wavelengths. Such continuous laser light sources are well known in the art.
As shown in fig. 1, an objective lens 9 is disposed on a side of the dichroic mirror 8 near the sample 10 to be measured, when microscopic imaging is performed on the sample 10 to be measured, the detection switching mirror 12 is moved between the dichroic mirror 8 and the photoelastic modulator 13, at this time, white light emitted by the white light source 11 sequentially passes through the sample 10 to be measured, the objective lens 9, and the dichroic mirror 8, and then is reflected by the detection switching mirror 12 to be injected into a microscopic imaging system, in this embodiment, the microscopic imaging system includes a fourth mirror 29, a second focusing lens 30, a CCD camera 31, and a second computer 33, where the light reflected by the detection switching mirror 12 is reflected again by the fourth mirror 29 and then is focused by the second focusing lens 30 to be injected into the CCD camera 31, and the CCD camera 31 is connected to the second computer 33 through a fifth data transmission line 32.
As shown in fig. 1, when the outgoing light of the switchable light source system is injected into the dichroic mirror 8 through the depolarizer 7, and the measured sample 10 is subjected to circular polarized fluorescence spectrum measurement, the detection switching mirror 12 moves out between the dichroic mirror 8 and the photoelastic modulator 13, the depolarizer 7 can depolarize the incident linear polarized excitation light beam into a natural excitation light beam with unpolarized characteristic, the wavelength of the natural excitation light beam is lower than the threshold wavelength of the dichroic mirror 8, when the natural excitation light beam is incident into the dichroic mirror 8, the dichroic mirror 8 reflects the natural excitation light beam to the measured sample 10, the measured sample 10 is excited by the natural excitation light beam and emits a circular polarized fluorescent light beam, the wavelength of the circular polarized fluorescent light beam is higher than the threshold wavelength of the dichroic mirror 8 and is transmitted through the dichroic mirror 8 and is incident into the photoelastic modulator 13, the photoelastic modulator 27 provides a periodically varying control signal to the photoelastic modulator 13, the phase of the photoelastic modulator 13 varies according to the periodically varying control signal, and the circular polarized fluorescent light beam is transmitted through the linear polarization modulator 13 to the selective polarization filter system, and the circular polarized fluorescent light beam is collected by the circular polarization modulator 14. Both the depolarizer 7 and the dichroic mirror 8 are well known in the art.
In this embodiment, as shown in fig. 2, the photoelastic modulator controller 27 sends out a periodic square wave signal, the square wave signal triggers the lattice rotation of the photoelastic modulator 13, the photoelastic modulator 13 is a quarter wave plate with a rotatable optical axis, the photoelastic modulator 13 is triggered by the rising edge of the square wave signal to rotate the optical axis, one signal period of the square wave signal is the optical axis rotation period, the rotation period is 180 °, the circularly polarized fluorescent light beam is converted into a linearly polarized fluorescent light beam after being transmitted through the photoelastic modulator 13, the polarization direction of the linearly polarized fluorescent light beam is parallel to the optical axis direction of the photoelastic modulator 13, the transmittance of the linearly polarized fluorescent light beam is highest when the polarization direction of the linearly polarized fluorescent light beam is 45 ° and 135 ° with the optical axis direction of the glaring prism 14 in the process of being transmitted through the glaring prism 14, the transmitted linearly polarized fluorescent light beam can obtain 2 times of strongest signal period, and the transmitted linearly polarized fluorescent light beam exhibits a light intensity of the angle of 45 ° with the circular polarization prism 14 when the circularly polarized fluorescent light beam is transmitted through the optical axis of the glaring prism 14, and the circularly polarized fluorescent light beam has a corresponding to the optical axis of 45 ° and the circular polarization of the circular prism 14. The photoelastic modulator 13, photoelastic modulator controller 27, and the gram prism 14 are all well known in the art, wherein the gram prism 14 may be secured to a frame that is mounted on a turntable having a Y-direction rotational degree of freedom.
As shown in fig. 1 and 3, the spectrum acquisition system includes a high-pass filter 15, a first focusing mirror 16, a light transmitting optical fiber 18, a concave reflecting mirror 19, a beam splitting grating 20, a photomultiplier 21, an acquisition board 23 and a first computer 25, the linear polarized fluorescent light beam screened by the grainy prism 14 is injected into an input end 17 of the light transmitting optical fiber 18 through the high-pass filter 15 and the first focusing mirror 16, wherein the excitation beam stray light possibly existing in the linear polarized fluorescent light beam is deleted by the high-pass filter 15, then the linear polarized fluorescent light beam with the stray light deleted is focused by the first focusing mirror 16 and then is injected into the input end 17 and transmitted and emitted to the concave reflecting mirror 19 through the light transmitting optical fiber 18, the light beam is collimated by the concave reflector 19 and is incident to the light splitting grating 20 to be widened and split in the wavelength direction to be incident to the photomultiplier 21, the photomultiplier 21 can convert an optical signal of the widened fluorescent light beam in a smaller wavelength range into an electric signal, in a rotation period of an optical axis of the photoelastic modulator 13, the photomultiplier 21 can convert the light intensity change of the fluorescent light beam in the period into an electric signal intensity change, the wavelength of the widened fluorescent light beam incident on the photomultiplier 21 is changed by rotating the light splitting grating 20 in the collection process, so that the fluorescence spectrum is completely collected, the photomultiplier 21 is connected with the collection board 23 through the first data transmission line 22, and the collection board 23 is connected with the first computer 25 through the second data transmission line. The light transmission optical fiber 18, the beam splitting grating 20, the photomultiplier 21 and the acquisition board card 23 are all well known in the art.
As shown in fig. 1, the photoelastic modulator controller 27 is connected to the collecting board 23 through a fourth data transmission line 28, the photoelastic modulator controller 27 not only provides a trigger signal to the photoelastic modulator 13, but also provides a trigger signal to the collecting board 23, the trigger signal provided to the collecting board 23 is a square wave signal, the collecting board 23 starts to collect the electrical signal converted by the photomultiplier 21 through the triggering of the rising edge of the square wave signal and transmits the electrical signal to the first computer 25, and the first computer 25 processes the information obtained by the collecting board 23 and displays the linear polarization fluorescence spectrum intensity through related software.
As shown in fig. 4, the bottom curve shows the linear polarization fluorescence spectrum corresponding to the left-hand circular polarization fluorescence beam of the sample 10 to be measured, wherein the abscissa is the wavelength, the ordinate is the linear polarization fluorescence spectrum relative light intensity of the corresponding wavelength, i.e. I L, and the middle (b) curve shows the difference between the left-hand circular polarization fluorescence beam spectrum relative light intensity (I L) and the right-hand circular polarization fluorescence beam spectrum relative light intensity (I R) of the sample 10 to be measured, i.e.:
Δ＝IL-IR；
The top (a) curve is the asymmetric coefficient of the fluorescence spectrum of the sample being measured, namely:
By analyzing the asymmetry coefficient, the circularly polarized fluorescence spectrum characteristic of the sample 10 to be measured can be obtained.
The working principle of the invention is as follows:
As shown in fig. 1, the present invention is provided with a light source switching mirror 4 between a first mirror 3 and a second mirror 5 of a switchable light source system, a detection switching mirror 12 between a dichroic mirror 8 and a photoelastic modulator 13, the light source switching mirror 4 being moved out of the system when a first continuous laser light source 1 is used as an excitation light source of the system, the light source switching mirror 4 being moved into the system when a second continuous laser light source 2 is used as an excitation light source of the system, thereby achieving switching of excitation light sources of different wavelengths. When the system carries out microscopic imaging focusing on the detected sample 10, the detection switching reflecting mirror 12 is moved into the system, and when the system carries out circular polarized fluorescence spectrum measurement on the detected sample 10, the detection switching reflecting mirror 12 is moved out of the system, so that different detection conversion is realized.
As shown in fig. 1, in the microscopic imaging process, the detection switching mirror 12 is moved into the system, white light emitted by the white light source 11 illuminates the sample 10 to be detected, and the light beam passing through the sample 10 to be detected sequentially passes through the objective lens 9, the dichroic mirror 8 and the detection switching mirror 12 and then is imaged in the microscopic imaging system, wherein the microscopic imaging system comprises a fourth mirror 29, a second focusing mirror 30, a CCD camera 31 and a second computer 33, the sample 10 to be detected is placed on a translation stage with three degrees of freedom of movement of X/Y/Z, and in the microscopic imaging process, the position of the sample 10 to be detected is adjusted by adjusting the translation stage, so that the sample 10 to be detected can form a clear and complete image on the CCD camera 31, and the second computer 33 can display the clear and complete image.
In addition, as shown in fig. 1, in the process of focusing the excitation light on the sample 10 to be tested, the detection switching mirror 12 is moved into the system, the sample 10 to be tested forms a clear and complete image on the CCD camera 31, the outgoing beam of the excitation light source of the switchable light source system is focused on the sample 10 to be tested, the focusing focus is displayed as a bright light spot on the second computer 33, the position of the bright light spot on the sample 10 to be tested is adjusted by adjusting the position angles of the second mirror 5 and the third mirror 6, so that the focusing of the excitation light on the sample 10 to be tested is completed, and in-situ measurement can be realized by the same method in repeated experiments.
As shown in fig. 1, in the process of collecting a circular polarized fluorescence spectrum of a sample 10 to be tested, a continuous laser light source with a target excitation wavelength in a switchable light source system is selected as an excitation light source according to requirements, a laser beam emitted by the excitation light source is depolarized into natural light by a depolarizer 7 and is subjected to selective focusing on the sample 10 to be tested, the circular polarized fluorescence beam emitted by the sample 10 to be tested is converted into a linear polarized fluorescence beam through a dichroic mirror 8, the linear polarized fluorescence beam is subjected to polarization selection by a grazing prism 14 and then is transmitted through a high-pass filter 15, and is focused to a spectrum collection system through a first focusing mirror 16, wherein the spectrum collection system comprises a light transmission optical fiber 18, a concave mirror 19, a beam splitting grating 20, a photomultiplier 21 and a collection board card 23, the spectrum collection system can convert the linear polarized fluorescence beam focused on an input end 17 of the light transmission optical fiber 18 into an electric signal, the electric signal strength and the linear polarized fluorescence beam strength are in a linear relation, and the electric signal strength is displayed after being processed by a first computer 25.
As shown in fig. 1, the spectrograting 20 in the spectrum acquisition system is placed on a turntable having a degree of freedom of rotation in the Z direction, and the wavelength of the linearly polarized fluorescent light beam incident on the photomultiplier tube 21 can be changed by changing the rotation angle of the turntable.
After the selected area of the measured sample is focused, circular polarization fluorescence spectrum measurement of the measured sample is performed.
In this embodiment, the light source switching mirror 4 and the detection switching mirror 12 have two degrees of automation, such as a degree of freedom of rotation that can be provided on a turntable, and a degree of freedom of movement that can be provided on a moving platform.
In this embodiment, the first continuous laser source 1 is preferably a continuous laser with a wavelength of 355nm, the second continuous laser source 2 is preferably a continuous laser with a wavelength of 405nm, the dichroic mirror 8 is preferably a critical wavelength of 410nm, the objective lens 9 is preferably a 200mm objective lens, the sample 10 to be measured is preferably a diamond anvil cell high-voltage module, the white light source 11 is preferably an LED white light source, and the high-pass filter 15 is preferably a critical wavelength of 430nm.
Claims (6)
1. A microscopic circular polarization fluorescence spectrum detection system based on a single photon counting method is characterized in that: the device comprises a switchable light source system, a depolarizer (7), a dichroic mirror (8), a white light source (11), a detection switching reflector (12), a photoelastic modulator (13), a graticule prism (14), a spectrum acquisition system and a microscopic imaging system, wherein the white light source (11), a tested sample (10), the dichroic mirror (8), the photoelastic modulator (13) and the graticule prism (14) are arranged in a line, the switchable light source system is arranged on one side of the dichroic mirror (8) and the emitted excitation light is emitted into the dichroic mirror (8) through the depolarizer (7), the detection switching reflector (12) is movably arranged between the dichroic mirror (8) and the photoelastic modulator (13), when the detection switching reflector (12) moves, the emitted light of the dichroic mirror (8) is reflected into the microscopic imaging system through the detection switching reflector (12), when the detection switching reflector (12) moves out, the emitted light of the dichroic mirror (8) passes through the photoelastic modulator (13) and the graticule prism (14), and the spectrum acquisition system (27) are connected with the spectrum acquisition system through the photoelastic modulator (27) through the line;
The switchable light source system comprises a first continuous laser light source (1), a second continuous laser light source (2), a first reflecting mirror (3), a second reflecting mirror (5), a third reflecting mirror (6) and a light source switching reflecting mirror (4), wherein the first reflecting mirror (3) is arranged on an output light path of the first continuous laser light source (1), the light source switching reflecting mirror (4) is arranged on an output light path of the second continuous laser light source (2) and is movably arranged between the first reflecting mirror (3) and the second reflecting mirror (5), when the light source switching reflecting mirror (4) moves out, emergent light of the first continuous laser light source (1) sequentially passes through the first reflecting mirror (3), the second reflecting mirror (5) and the third reflecting mirror (6) to be reflected into the depolarizer (7), and when the light source switching reflecting mirror (4) moves in, emergent light of the second continuous laser light source (2) sequentially passes through the light source switching reflecting mirror (4), the second reflecting mirror (5) and the third reflecting mirror (6) to be reflected into the depolarizer (7);
The photoelastic modulator (13) sends out a periodical signal through the photoelastic modulator controller (27) to control periodical rotation, and the linear polarized fluorescent light beam formed after passing through the photoelastic modulator (13) and the graticule prism (14) obtains 2 times of strongest light beam intensity in one period, wherein one time is the linear polarized fluorescent light beam formed by the left-hand circular polarized fluorescent light beam, the other time is the linear polarized fluorescent light beam formed by the right-hand circular polarized fluorescent light beam, and the spectrum acquisition system acquires the linear polarized fluorescent light beam and calculates and obtains the relative light intensity of the left-hand circular polarized fluorescent light beam spectrum Relative light intensity to right-hand circularly polarized fluorescent light beam spectrumThe difference is:
；
Meanwhile, the fluorescence spectrum asymmetry coefficient of the tested sample (10) is obtained, namely:
；
obtaining circular polarized fluorescence spectrum characteristics of the sample (10) to be tested through analysis of the asymmetry coefficient;
the photoelastic modulator controller (27) emits a periodic square wave signal and the photoelastic modulator (13) is triggered by the rising edge of the square wave signal.
2. The single photon counting method-based microscopic circular polarized fluorescence spectrum detection system of claim 1, wherein: the microscopic imaging system comprises a fourth reflecting mirror (29), a second focusing lens (30), a CCD camera (31) and a second computer (33), wherein light rays reflected by the detection switching reflecting mirror (12) are reflected again by the fourth reflecting mirror (29) and then focused and injected into the CCD camera (31) through the second focusing lens (30), and the CCD camera (31) is connected with the second computer (33) through a circuit.
3. The single photon counting method-based microscopic circular polarized fluorescence spectrum detection system of claim 1, wherein: the spectrum acquisition system comprises a high-pass filter (15), a first focusing lens (16), a light transmission optical fiber (18), a concave reflecting mirror (19), a beam splitting grating (20), a photomultiplier (21), an acquisition board card (23) and a first computer (25), wherein light beams screened by a gram prism (14) are injected into the light transmission optical fiber (18) through the high-pass filter (15) and the first focusing lens (16), are transmitted to the concave reflecting mirror (19) through the light transmission optical fiber (18), are reflected to the beam splitting grating (20) through the concave reflecting mirror (19), are split into the photomultiplier (21) through the beam splitting grating (20), the photomultiplier (21) is connected with the acquisition board card (23) through a circuit, and the acquisition board card (23) is connected with the first computer (25) through the circuit.
4. A single photon counting method based microscopic circular polarized fluorescence spectrum detection system according to claim 3, wherein: the photoelastic modulator controller (27) is connected with the acquisition board card (23) through a circuit.
5. The single photon counting method-based microscopic circular polarized fluorescence spectrum detection system of claim 1, wherein: one signal period of the square wave signal is one rotation period of the optical axis of the photoelastic modulator (13), and the rotation period is 180 °.
6. The single photon counting method-based microscopic circular polarized fluorescence spectrum detection system of claim 5, wherein: the circular polarized fluorescent light beams corresponding to the optical axes of the photoelastic modulator (13) and the optical axes of the Greenwich prisms (14) are left-handed circular polarized fluorescent light beams when the included angles of the optical axes of the photoelastic modulator (13) and the optical axes of the Greenwich prisms (14) are 45 degrees, and the circular polarized fluorescent light beams corresponding to the optical axes of the photoelastic modulator (13) and the optical axes of the Greenwich prisms (14) are right-handed circular polarized fluorescent light beams.
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