CN116698740A - Microscopic linear polarization fluorescence spectrum imaging measurement system - Google Patents
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Abstract
The invention belongs to the field of optical detection of samples, and particularly relates to a microscopic linear polarization fluorescence spectrum imaging measurement system. Comprising the following steps: the device comprises a reflecting mirror II, a reflecting objective I, a reflecting objective II, a movable reflecting mirror, a 1/2 wave plate, a Greenland prism, a high-pass filter, a focusing mirror I, a light source generating system, a microscopic imaging system and a spectrum acquisition system which are arranged in sequence in a line; the light source generation system is arranged on an incident light path of the second reflecting mirror; the microscopic imaging system is arranged on a reflecting light path of the movable reflecting mirror; the spectrum acquisition system is arranged on a transmission light path of the first focusing lens; and a space for placing a sample to be measured is arranged between the first reflective objective lens and the second reflective objective lens. The proposal of the invention for collecting and measuring the fluorescence at different positions of the sample area of the measured sample enables accurate measurement of the linear polarization fluorescence spectrum of the measured sample and collection of the linear polarization fluorescence spectrum at different positions of the measured sample to be possible.
Description
Technical Field
The invention belongs to the field of optical detection of samples, and particularly relates to a microscopic linear polarization fluorescence spectrum imaging measurement system.
Background
The linear polarization fluorescence spectrum measurement system is a device which uses linear polarization excitation light to excite a characteristic spectrum of sample molecules and collects and displays linear polarization fluorescence radiated by a sample to be measured, and the measurement of the linear polarization fluorescence spectrum can assist in analyzing chemical characteristics of the molecules.
In the process of performing linear polarization fluorescence spectrum measurement, in order to complete excitation of excitation light on a sample to be measured and collection of linear polarization fluorescence of the sample to be measured on the same side of the sample to be measured, a dichroic mirror is generally used to realize separation of the linear polarization fluorescence and the excitation light, and due to different transmissivity of the dichroic mirror to linear polarization light beams in different directions, actual linear polarization fluorescence of the sample cannot be accurately collected.
For a sample to be tested with uneven spatial distribution, particularly a diamond anvil cell high-pressure sample and a liquid nitrogen or liquid helium cooled low-temperature sample, the linear polarization fluorescence of the radiation of different positions of the sample after being excited by the linear polarization excitation light is different, so that it is necessary to accurately collect the linear polarization fluorescence spectrum information of different positions of the sample, and no related achievement report capable of realizing the function exists at present.
Disclosure of Invention
In order to solve the problems, the invention provides a scheme capable of accurately collecting and measuring the linear polarized fluorescence of the measured sample and collecting and measuring the fluorescence of different positions of a sample area of the measured sample, so that the accurate measurement of the linear polarized fluorescence spectrum of the measured sample and the collection of the linear polarized fluorescence spectrum of different positions of the measured sample are possible.
The invention aims to provide a linear polarization fluorescence spectrum imaging measurement system, which divides a sample area of a sample to be measured into a plurality of rows and columns of matrixes, uses linear polarization excitation light to excite the sample area corresponding to a corresponding matrix element, collects linear polarization fluorescence radiated by the sample area by a spectrometer, and completes the collection of the linear polarization fluorescence spectrum of the matrix element position corresponding to the sample area by changing the position of the sample to be measured, thereby realizing the imaging measurement of the linear polarization fluorescence spectrum of the sample to be measured.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a microscopic linear polarized fluorescence spectroscopy imaging measurement system comprising: the device comprises a reflecting mirror II, a reflecting objective I, a reflecting objective II, a movable reflecting mirror, a 1/2 wave plate, a Greenland prism, a high-pass filter, a focusing mirror I, a light source generating system, a microscopic imaging system and a spectrum acquisition system which are arranged in sequence in a line;
the light source generation system is arranged on an incident light path of the second reflecting mirror; the microscopic imaging system is arranged on a reflecting light path of the movable reflecting mirror; the spectrum acquisition system is arranged on a transmission light path of the first focusing lens; and a space for placing a sample to be measured is arranged between the first reflective objective lens and the second reflective objective lens.
The light source generating system includes: the movable beam splitting sheet, the polarizing plate and the first reflecting mirror are sequentially arranged on the incident light path of the second reflecting mirror in a line, and the movable beam splitting sheet, the polarizing plate and the first reflecting mirror further comprise a laser light source arranged on the incident light path of the first reflecting mirror and a white light source arranged on the incident light path of the movable beam splitting sheet.
The polarizer is provided on a mirror holder having a degree of freedom of rotation about the laser propagation direction.
The microscopic imaging system includes: the CCD camera is connected with the second computer through the second data transmission line, and the third reflecting mirror, the second focusing mirror and the CCD camera are sequentially arranged on a reflecting light path of the movable reflecting mirror.
The microscopic imaging system includes: the optical fiber transmission system comprises an optical fiber input end, an optical fiber, a spectrometer, a first data transmission line and a first computer which are connected in sequence, wherein the optical fiber input end is arranged on a transmission light path of a first focusing lens.
The light transmission optical fiber input end is arranged on the lens frame with the freedom degree of movement in the vertical direction and the light path propagation direction.
When the microscopic imaging is carried out on the sample to be detected, the movable beam splitting piece moves to a position between the polarizing plate and the reflecting mirror II, the movable reflecting mirror is positioned between the reflecting objective II and the 1/2 wave plate, and white light emitted by the white light source sequentially passes through the movable beam splitting piece, the reflecting mirror II and the reflecting objective to illuminate the sample to be detected, so that the sample to be detected sequentially passes through the reflecting objective II and the movable reflecting mirror to be imaged in the microscopic imaging system.
When the spectrum measurement is carried out on the measured sample, the movable beam splitting piece and the movable reflecting mirror are respectively moved out of the linear arrangement, natural excitation light beams emitted by the laser light source sequentially pass through the first reflecting mirror, the second polarizing plate, the second reflecting mirror of the array, the first reflecting objective lens and irradiate on the measured sample, so that the measured sample is excited to radiate linear polarized fluorescence, and the linear polarized fluorescence sequentially passes through the second reflecting objective lens, the 1/2 wave plate, the Greenwich prism, the high-pass filter and the first focusing mirror to carry out the spectrum measurement in the spectrum acquisition system.
The sample to be measured is fixed on a translation stage with freedom of movement in the XYZ direction.
The 1/2 wave plate is arranged on the electric lens frame with the freedom degree of rotation around the light beam propagation direction.
The microscopic linear polarization fluorescence spectrum imaging measurement method comprises the following steps:
when the microscopic imaging is carried out on the sample to be detected, the movable beam splitting piece moves between the polarizing plate and the reflecting mirror II, the movable reflecting mirror is positioned between the reflecting objective II and the 1/2 wave plate, and white light emitted by the white light source sequentially passes through the movable beam splitting piece, the reflecting mirror II and the reflecting objective to illuminate the sample to be detected, so that the sample to be detected sequentially passes through the reflecting objective II and the movable reflecting mirror to be imaged in the microscopic imaging system;
when the spectrum measurement is carried out on the measured sample, the movable beam splitting piece and the movable reflecting mirror are respectively moved out of the linear arrangement, natural excitation light beams emitted by the laser light source sequentially pass through the first reflecting mirror, the second polarizing plate, the second reflecting mirror of the array, the first reflecting objective lens and irradiate on the measured sample, so that the measured sample is excited to radiate linear polarized fluorescence, and the linear polarized fluorescence sequentially passes through the second reflecting objective lens, the 1/2 wave plate, the Greenwich prism, the high-pass filter and the first focusing mirror to carry out the spectrum measurement in the spectrum acquisition system.
The invention has the following beneficial effects and advantages:
1. the invention is provided with the movable beam splitting sheet and the movable reflecting mirror, when the movable beam splitting sheet moves between the polarizing sheet and the second reflecting mirror and the movable reflecting mirror moves between the second reflecting objective lens and the 1/2 wave plate, the white light source and the microscopic imaging system are switched into the light path, and the system can perform microscopic imaging and selective focusing on a sample to be tested, so that in-situ measurement can be performed on the same position of the sample to be tested in the repeated experiment process.
2. The invention is provided with the reflective objective I and the reflective objective II, creatively removes the dichroic mirror in the similar report, leads the incident direction of the laser light source and the collecting direction of the linear polarized fluorescent light beam of the tested sample to be respectively positioned at two sides of the tested sample, leads the fluorescent spectrum collected by the spectrum collecting system to be consistent with the fluorescent spectrum of the radiation of the tested sample, and eliminates the problem that the finally collected linear polarized fluorescent polarization component and the linear polarized fluorescent polarization component of the radiation of the sample are inconsistent due to the fact that the dichroic mirror is adopted for carrying out light splitting when the incident direction of the laser light source is consistent with the collecting direction of the linear polarized fluorescent light beam of the tested sample.
3. The invention is characterized in that the measured sample is arranged on a three-dimensional translation stage driven by a linear motor, the three-dimensional translation stage is driven by the linear motor to move in the measuring process, the superposition position of the measured sample and the laser source beam is changed, and the system collects the linear polarized fluorescence radiated by different positions of the measured sample and corresponds the linear polarized fluorescence to the excited position of the measured sample, thereby realizing the imaging measurement of the linear polarized fluorescence spectrum of the measured sample.
Drawings
FIG. 1 is a schematic diagram of the structure of the present invention;
FIG. 2 is a schematic diagram of a selective focusing mode according to the present invention;
FIG. 3 is a schematic diagram of a spectroscopic imaging measurement method of the present invention;
wherein 1 is a laser light source, 2 is a white light source, 3 is a reflector I, 4 is a polaroid, 5 is a movable beam splitter, 6 is a reflector II, 7 is a reflective objective I, 8 is a sample to be measured, 9 is a reflective objective II, 10 is a movable reflector, 11 is a 1/2 wave plate, 12 is a gram prism, 13 is a high-pass filter, 14 is a focusing mirror I, 15 is a light transmitting optical fiber input end, 16 is a light transmitting optical fiber, 17 is a spectrometer, 18 is a first data transmission line, 19 is a first computer, 20 is a reflector III, 21 is a focusing mirror II, 22 is a CCD camera, 23 is a second data transmission line, and 24 is a second computer.
Where 801 is the sample 8 to be measured imaged in the microscopic imaging system, and 101 is the sampling matrix of the laser light source 1 or the laser light source 2 focusing the coincident light spot on the sample 8 to be measured during imaging measurement.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
The linear polarization fluorescence spectrum imaging measurement system comprises a laser light source, a white light source, a first reflecting mirror, a movable beam splitter, a second reflecting mirror, a first reflecting object lens, a measured sample, a second reflecting mirror, a movable reflecting mirror, a 1/2 wave plate, a gram prism, a high-pass filter, a first focusing mirror, a spectrum acquisition system and a microscopic imaging system, wherein the first reflecting mirror, the measured sample, the second reflecting mirror, the movable reflecting mirror, the 1/2 wave plate, the gram prism, the high-pass filter, the focusing mirror and the spectrum acquisition system are arranged in a line, when the microscopic imaging is carried out on the measured sample, the movable beam splitter is moved between the polarizing plate and the second reflecting mirror, the movable reflecting mirror is moved between the second reflecting mirror and the first reflecting mirror, white light emitted by the white light source is illuminated by the measured sample after passing through the movable beam splitter, the second reflecting mirror and the first reflecting mirror, the measured sample is imaged in the microscopic imaging system, when the movable beam splitter and the movable reflecting mirror are polarized and the linear fluorescence light are carried out on the measured sample, the movable beam splitter and the movable reflecting mirror are sequentially moved out on the first reflecting mirror, the linear light is emitted by the reflecting mirror and the first reflecting mirror and the linear light is excited by the reflecting mirror, the linear light is acquired by the reflecting mirror and the first reflecting mirror and the linear light is polarized by the reflecting mirror, and the linear light is sequentially reflected by the reflecting mirror and the linear light is acquired by the reflecting mirror.
The first reflective objective lens may be replaced by a focusing system.
The movable beam splitting piece and the movable reflecting mirror are respectively provided with a moving-in system and a moving-out system, and the two states are realized through an electric overturning platform.
The polarizing plates and the graticule prisms are respectively mounted on a frame having a degree of freedom of rotation in a direction in which a light beam propagates.
The 1/2 wave plate is arranged on an electric lens frame with a rotational freedom degree in the light beam propagation direction, and the rotation of the electric lens frame can drive the rotation of the 1/2 wave plate.
The sample to be measured is mounted on a translation stage having a degree of freedom of movement in the direction X, Y, Z, the degree of freedom of movement of the translation stage being driven by a linear motor.
The microscopic imaging system consists of a third reflecting mirror, a second focusing mirror, an imaging camera, a second data transmission line and a second computer.
The spectrum acquisition system consists of a light transmission optical fiber, a spectrometer, a first data transmission line and a first computer, wherein the incident section and the emergent end of the light transmission optical fiber are provided with optical fiber ends.
The sample to be tested can be a spin-coated solid sample, can be a diamond anvil cell high-pressure module, and can be a liquid nitrogen or liquid helium refrigerated low-temperature sample.
As shown in fig. 1, the invention comprises a laser light source 1, a white light source 2, a reflecting mirror 3, a reflecting mirror 1, a reflecting mirror 4, a polarizing plate 5, a movable beam splitter plate 6, a reflecting mirror 7, a reflecting object lens 1, a reflecting object lens 8, a measured sample 9, a reflecting object lens 10, a movable reflecting mirror 11, a reflecting mirror 1/2, a reflecting mirror 12, a graniser prism 13, a high-pass filter plate, a spectrum acquisition system and a microscopic imaging system, wherein the reflecting mirror 6, the reflecting object lens 7, the measured sample 8, the reflecting object lens 9, the movable reflecting mirror 10, the 1/2 wave plate 11, the graniser prism 12, the high-pass filter plate 13, the focusing mirror 14 and the spectrum acquisition system are arranged in a line, when microscopic imaging is carried out on the measured sample 8, the movable beam splitter plate 5 moves between the polarizing plate 4 and the reflecting mirror 6, white light emitted by the light source 2 sequentially passes through the movable beam splitter plate 5, the reflecting mirror 6 and the reflecting object lens 7, the measured sample 8 is illuminated by the reflecting mirror 8, the measured sample 8 is imaged by the reflecting mirror 9, the reflecting mirror 10 can be imaged by the reflecting mirror 1/2, the laser light beam is reflected by the reflecting mirror 1/2, the reflecting mirror 1/the laser beam is reflected by the reflecting mirror 1, the fluorescent light is reflected by the reflecting mirror 1 and the reflecting mirror 4, and the fluorescent light is reflected by the reflecting mirror 1, and the fluorescent light is reflected by the reflecting mirror 4 is reflected by the reflecting mirror 6, and the fluorescent light is reflected by the reflecting mirror 4.
As shown in fig. 2, in microscopic imaging, a sample 8 to be measured is imaged in a microscopic imaging system, 801 is that the sample 8 to be measured is imaged in the microscopic imaging system, laser emitted by the laser source 1 sequentially passes through the polarizing plate 4, the movable beam splitting plate 5, the reflecting mirror two 6 and the reflecting objective lens 7, and then is focused on the sample 8 to be measured to generate a focused coincident light spot, the focused coincident light spot can be imaged in the microscopic imaging system, 101 is that the laser source 1 focuses coincident light spot on the sample 8 to be imaged in the microscopic imaging system, and by adjusting the position of the sample 8 to be measured perpendicular to the incident direction of the laser, the coincident position of the focused coincident light spot and the sample area of the sample 8 to be measured can be changed, and the coincident position of the sample 8 to be measured and the laser is changed, so that the coincident position of the sample 8 to be measured in multiple measurements is the same, and thus in-situ measurement in multiple repeated measurement processes is realized.
As shown in fig. 3, during linear polarization fluorescence spectrum imaging measurement, a sample 8 is imaged in a microscopic imaging system, a laser light source 801 is formed by imaging the sample 8 in the microscopic imaging system, laser light emitted by the laser light source 1 sequentially passes through a polarizer 4, a movable beam splitter 5, a reflecting mirror two 6 and a reflecting objective lens 7, and then is focused on the sample 8 to generate a focused coincident light spot, the focused coincident light spot can also be imaged in the microscopic imaging system, each element in 101 is formed by imaging the focused coincident light spot on the sample 8 by the laser light source 1 during single acquisition in the linear polarization fluorescence spectrum imaging measurement process in the microscopic imaging system, the sample 8 is fixed on a translation stage with an XYZ-direction movement degree of freedom, the XYZ-direction movement degree of the translation stage is driven by a linear motor, the linear motor can control a movement step length of the translation stage, so that the sample 8 fixed on the translation stage moves in a direction perpendicular to the excitation light, and the fixed step length forms a linear polarization spectrum measurement matrix, namely, the linear polarization spectrum measurement is performed at each node.
The working principle of the invention is as follows:
as shown in fig. 1, the present invention is provided with a movable beam splitter 5 and a movable mirror 10, wherein the movable beam splitter 5 is moved between a polarizer 4 and a second mirror 6, and when the movable mirror 10 is moved between the second reflective objective lens 9 and a 1/2 wave plate 11, the white light source 2 and the microscopic imaging system are switched into the optical path, and when the linear polarized fluorescence spectrum measurement is performed on the sample 8 to be measured, the movable beam splitter 5 and the movable mirror 10 are removed from the system.
As shown in fig. 1, in the microscopic imaging process, the movable beam splitter 5 is moved between the polarizing plate 4 and the second reflecting mirror 6, the movable reflecting mirror 10 is moved between the second reflecting objective 9 and the 1/2 wave plate 11, white light emitted by the white light source 2 sequentially passes through the movable beam splitter 5, the second reflecting mirror 6 and the reflecting objective 7 to illuminate the sample 8 to be measured, the sample 8 to be measured passes through the second reflecting objective 9 and the movable reflecting mirror 10 and then is imaged in a microscopic imaging system, the microscopic imaging system is composed of the second focusing mirror 21, the CCD camera 22, the second data transmission line 23 and the second computer 24, the sample 8 to be measured is placed on a translation stage with X, Y, Z three degrees of freedom, in the microscopic imaging process, the position of the sample 8 to be measured is adjusted by adjusting the translation stage to translate the adjusting rod in X, Y, Z directions, the sample 8 to be measured can form a clear and complete image on the CCD camera 22, and the second computer 24 receives the imaging signal transmitted by the second data transmission line 23 to display the complete image.
As shown in fig. 2, in the selective focusing process, while the sample 8 to be measured is imaged in the microscopic imaging system, the laser emitted by the laser source 1 sequentially passes through the first reflecting mirror 3, the polarizing plate 4, the movable beam splitting plate 5, the second reflecting mirror 6 and the first reflecting objective 7 and then is focused on the sample area of the sample 8 to be measured, the laser forms a focusing coincident light spot at the focusing coincident position of the sample 8 to be measured, the focusing coincident light spot sequentially passes through the second reflecting objective 9 and the movable reflecting mirror and then is imaged in the microscopic imaging system, 801 is imaged by the sample 8 to be measured in the microscopic imaging system, 101 is imaged by the laser source 1 on the sample 8 to be measured, in the single measuring process, the coincident position of the sample 8 to be measured and the laser is changed by adjusting the position of the sample 8 to be measured perpendicular to the incident direction of the laser, the coincident position of the sample 8 to be measured is changed, in the repeated measuring process is changed by adjusting the position of the sample 8 to be measured perpendicular to the incident direction of the laser, and in the repeated measuring process is repeated with the laser in the repeated measuring process.
As shown in fig. 1, in the process of measuring the linear polarization fluorescence spectrum, a movable beam splitter 5 and a movable reflecting mirror 10 are moved out of the system, laser emitted from a laser light source 1 is reflected by a reflecting mirror 3 and then enters a polarizing plate 4, the polarizing plate 4 is arranged on a lens holder with a degree of freedom of rotation around the propagation direction of the laser, the polarization direction of the polarizing plate 4 is changed by adjusting the lens holder, the laser becomes pure linear polarization laser after passing through the polarizing plate 4, the pure linear polarization laser is focused on a sample 8 to be measured after passing through a reflecting mirror 6 and a reflecting objective 7 in sequence, the sample 8 to be measured is excited by the pure linear polarization laser and then emits linear polarization fluorescence, the linear polarization fluorescence beam consists of linear polarization beams with mutually perpendicular polarization directions, the linear polarized fluorescence is collimated into parallel light beams by a reflective objective lens 9 and then enters a 1/2 wave plate 11, the 1/2 wave plate 11 is arranged on an electric rotating frame with a rotation degree of freedom in the direction of light beam propagation, the electric rotating frame is set to rotate at a 0 DEG position and a 45 DEG position, a gram prism 12 is matched with the 0 DEG position and the 45 DEG position to respectively extract two polarized light beams of the linear polarized fluorescence light beams with mutually perpendicular polarization directions, the two polarized light beams respectively enter a spectrum acquisition system with the 0 DEG position and the 45 DEG position of the electric rotating frame, the linear polarized fluorescence light beams extracted by the gram prism are focused on a light transmission optical fiber input end 15 of a light transmission optical fiber 16 by a high-pass filter 13 and a focusing lens 14, the light transmission optical fiber input end 15 is arranged on the frame with a movement degree of freedom in the vertical and light path propagation directions, the position of the light transmission optical fiber input end 15 is changed by adjusting the mirror holder with the freedom of movement, so that the linear polarization fluorescent light beam has the maximum coupling efficiency on the light transmission optical fiber input end 15, the linear polarization fluorescent light beam is incident to the light transmission optical fiber input end 15 and is transmitted to the spectrometer 17 through the light transmission optical fiber 16, the spectrometer 17 collects and obtains the spectrum information of the linear polarization fluorescent light beam and converts the spectrum information into an electric signal, the telecommunication signal is transmitted to the first computer 19 through the first data transmission head 18, and the first computer displays and stores the electric signal converted by the linear polarization fluorescent light spectrum.
In the linear polarized fluorescence spectrum imaging process, a sample 8 to be measured is fixed on a three-dimensional translation stage with XYZ-direction movement freedom, the movement freedom of the translation stage is driven by a linear motor, microscopic imaging of the sample 8 to be measured is carried out before the imaging measurement process, the initial position of the imaging process is determined, namely, the superposition position of a sample area of the sample 8 to be measured and an excitation light focus is determined, a first group of linear polarized fluorescence spectrums are acquired after the initial position is determined, after the linear polarized fluorescence spectrums of the initial position are acquired, the three-dimensional translation position is changed through the linear motor, then the superposition position of the sample 8 to be measured and the excitation light focus is changed, the distance between the changed sample position and the initial position is a movement step length, the linear polarized fluorescence spectrums after the position change are continuously acquired, the position of the step length is repeatedly changed, the operation of acquiring the linear polarized fluorescence spectrums is carried out, the acquisition position covers the sample area of the sample 8 to be measured in a matrix form, and a linear polarized fluorescence spectrum imaging chart of the sample 8 to be measured can be synthesized through software after the linear polarized fluorescence spectrums of all matrix element positions are acquired.
After microscopic imaging and selective focusing of the measured sample 8 are completed, linear polarization fluorescence spectrum measurement of the measured sample 8 is carried out, and linear polarization fluorescence spectrum measurement is carried out on the measured sample 8 at different positions to realize linear polarization fluorescence spectrum imaging measurement.
In this embodiment, the first reflective objective lens 7 and the second reflective objective lens 9 are preferably 15 times of the objective lens, and the first focusing lens 14 is preferably 100mm in focal length.
Claims (10)
1. A microscopic linear polarized fluorescence spectroscopy imaging measurement system, comprising: the device comprises a second reflecting mirror (6), a first reflecting objective (7), a second reflecting objective (9), a movable reflecting mirror (10), a 1/2 wave plate (11), a Grignard prism (12), a high-pass filter (13), a first focusing mirror (14) which are sequentially arranged in a line, and further comprises a light source generation system, a microscopic imaging system and a spectrum acquisition system;
the light source generation system is arranged on an incident light path of the second reflecting mirror (6); the microscopic imaging system is arranged on a reflecting light path of the movable reflecting mirror (10); the spectrum acquisition system is arranged on a transmission light path of the first focusing lens (14); a space for placing a sample (8) to be measured is arranged between the first reflective objective (7) and the second reflective objective (9).
2. The microscopic linear polarized fluorescence spectroscopy imaging measurement system of claim 1, wherein the light source generation system comprises: the movable beam splitting sheet (5), the polarizing sheet (4) and the reflecting mirror I (3) which are arranged in a line on the incident light path of the reflecting mirror II (6) in sequence, and the movable beam splitting sheet also comprises a laser light source (1) arranged on the incident light path of the reflecting mirror I (3) and a white light source (2) arranged on the incident light path of the movable beam splitting sheet (5).
3. The microscopic linear polarized fluorescence spectroscopy imaging measurement system of claim 2, wherein the polarizer (4) is provided on a frame having rotational freedom about the laser propagation direction.
4. The microscopic linear polarized fluorescence spectroscopy imaging measurement system of claim 1, wherein the microscopic imaging system comprises: the device comprises a reflector III (20), a focusing mirror II (21), a CCD camera (22), a second data transmission line (23) and a second computer (24), wherein the CCD camera (22) is connected with the second computer (24) through the second data transmission line (23), and the reflector III (20), the focusing mirror II (21) and the CCD camera (22) are sequentially arranged on a reflecting light path of the movable reflector (10).
5. The microscopic linear polarized fluorescence spectroscopy imaging measurement system of claim 1, wherein the microscopic imaging system comprises: the optical fiber focusing device comprises an optical fiber input end (15), an optical fiber (16), a spectrometer (17), a first data transmission line (18) and a first computer (19) which are connected in sequence, wherein the optical fiber input end (15) is arranged on a transmission light path of a focusing mirror I (14).
6. The system according to claim 5, wherein the light-transmitting fiber input end (15) is mounted on a frame having freedom of movement in the direction perpendicular to the direction of propagation of the light path.
7. The microscopic linear polarization fluorescence spectrum imaging measurement system according to claim 1 or 2, wherein when the microscopic imaging is performed on the sample to be measured, the movable beam splitter (5) is moved between the polarizer (4) and the second reflecting mirror (6), the movable reflecting mirror (10) is positioned between the second reflecting objective (9) and the 1/2 wave plate (11), and white light emitted by the white light source (2) sequentially passes through the movable beam splitter (5), the second reflecting mirror (6) and the reflecting objective (7) to back-illuminate the sample to be measured (8), so that the sample to be measured (8) sequentially passes through the second reflecting objective (9) and the movable reflecting mirror (10) to be imaged in the microscopic imaging system.
8. The microscopic linear polarization fluorescence spectrum imaging measurement system according to claim 1 or 2, wherein when the measured sample is subjected to spectrum measurement, the movable beam splitter (5) and the movable reflecting mirror (10) are respectively moved out of alignment, natural excitation light beams emitted by the laser light source (1) sequentially pass through the reflecting mirror I (3), the polarizing plate (4), the reflecting mirror II (6) of the row, the reflecting objective I (7) and irradiate on the measured sample (8), so that the measured sample (8) is excited and then radiates linear polarization fluorescence, and the linear polarization fluorescence passes through the reflecting objective II (9), the 1/2 wave plate (11), the gram prism (12), the high-pass filter (13) and the focusing mirror I (14) to be subjected to spectrum measurement in the spectrum acquisition system.
9. The microscopic linear polarized fluorescence spectroscopy imaging measurement system of claim 1, wherein the sample under test (8) is fixed on a translation stage having XYZ-direction movement degrees of freedom; the 1/2 wave plate (11) is arranged on an electric lens holder with the freedom of rotating around the light beam propagation direction.
10. The microscopic linear polarization fluorescence spectrum imaging measurement method is characterized by comprising the following steps of:
when a sample to be detected is subjected to microscopic imaging, the movable beam splitting sheet (5) moves between the polarizing sheet (4) and the reflecting mirror II (6), the movable reflecting mirror (10) is positioned between the reflecting objective II (9) and the 1/2 wave plate (11), and white light emitted by the white light source (11) sequentially passes through the movable beam splitting sheet (5), the reflecting mirror II (6) and the reflecting objective (7) to illuminate the sample to be detected (8), so that the sample to be detected (8) sequentially passes through the reflecting objective II (9) and the movable reflecting mirror (10) and then is imaged in a microscopic imaging system;
when the spectrum measurement is carried out on the measured sample, the movable beam splitting sheet (5) and the movable reflecting mirror (10) are respectively moved out of a linear arrangement, natural excitation light beams emitted by the laser light source (1) sequentially pass through the reflecting mirror I (3), the polarizing sheet (4), the reflecting mirror II (6) of the row, the reflecting objective lens I (7) and irradiate on the measured sample (8), so that the excited radiation of the measured sample (10) is linear polarized fluorescence, and the excited radiation is sequentially passed through the reflecting objective lens II (9), the 1/2 wave plate (11), the grazing prism (12), the high-pass filter (13) and the focusing mirror I (14) to carry out spectrum measurement in a spectrum acquisition system.
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| CN (1) | CN116698740A (en) |
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2022
- 2022-02-24 CN CN202210171481.7A patent/CN116698740A/en active Pending
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