CN117250178A - Fluorescence anisotropy detection method and detection system thereof - Google Patents
Fluorescence anisotropy detection method and detection system thereof Download PDFInfo
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- 238000000198 fluorescence anisotropy Methods 0.000 title claims abstract description 49
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- 238000002073 fluorescence micrograph Methods 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 231100000324 minimal toxicity Toxicity 0.000 description 1
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- 210000000056 organ Anatomy 0.000 description 1
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- 150000003384 small molecules Chemical class 0.000 description 1
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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 discloses a fluorescence anisotropy detection method and a detection system thereof, wherein the system comprises a polarized laser transmitter, a collimating and beam-expanding lens group and a polarization beam splitter, wherein the polarized laser transmitter is used for transmitting polarized laser; one end of the polarization beam splitter is provided with a first half-wave plate and a stripe modulator which are coaxially arranged, the other end of the polarization beam splitter is provided with a lens, and the tail end of the lens is provided with a variable-pitch spatial filter, a partition half-wave plate and a relay lens which are coaxially arranged; the end of the relay lens is sequentially provided with a dichroic mirror, an objective lens and a sample; an emission filter is arranged on one side of the dichroic mirror, the axis of the emission filter is perpendicular to the axis of the objective lens, and a second half-wave plate, a tube lens and a Wollaston polarizing prism are coaxially arranged at the tail end of the emission filter; the end of the Wollaston polarizing prism is provided with a light beam adjusting mirror group, and the end of the light beam adjusting mirror group is provided with a camera. The invention can realize the detection of fluorescence anisotropy by combining the detection method of the system.
Description
Technical Field
The invention relates to the technical field of structured light microscopic imaging, in particular to a fluorescence anisotropy detection method and a detection system thereof.
Background
In order for a drug to be clinically successful, it must produce the desired therapeutic effect without toxicity or with minimal toxicity and acceptability. In order to better understand the in vivo drug effect, it is necessary to directly measure the effect of targeted drugs in single cells and in cell populations that make up tissues and organs. Fluorescent labeling microscopy is widely applied to observation of subcellular structures of biological samples. The dipole orientation of fluorescent molecules can reflect the spatial orientation of the target protein, and further study the structure and kinetic problems of the target protein in living cells. Fluorescent molecules preferentially absorb dipoles that are parallel to the electric vector. When polarized light is projected onto the molecules, the molecules in the polarization direction preferentially absorb fluorescence, and selectively excite the fluorescent molecules in the dipole arrangement. The relative angle formed between the fluorescence absorption and the emission polarization is the fluorescence anisotropy (Fluorescence Anisotropy, FA).
Fluorescence anisotropy fluorescence-based techniques have highlighted an important role in the field of pharmaceutical research. The principle of fluorescence anisotropy is that at constant temperature and solution viscosity, the degree of anisotropy of a fluorophore is inversely proportional to its molecular rotation. Fluorescence anisotropy is exhibited more when fluorescent molecules bind or diffuse and spin more slowly in a high viscosity medium. When fluorescent molecules spin diffuse or resonate causing energy transfer, thereby reducing polarization, i.e. exhibiting a low fluorescence anisotropy. By reading the fluorescence anisotropy value of the small molecule, the binding and dissociation constants between the target and the fluorescently labeled drug can be measured.
In order to realize the measurement of the FA, the prior art provides an FA measurement imaging method based on fluorescence imaging. The technology realizes the detection of fluorescence anisotropy by constructing a fluorescence anisotropy parameter model through fluorescence excitation in the polarization direction and fluorescence imaging in the detection orthogonal polarization direction. However, it does not enable high-speed, dynamic structured light illumination imaging. In order to realize the structured light imaging, the second prior art proposes the structured light tomography technology. The technology realizes the tomography of fluorescent samples through high-frequency signal extraction by the characteristic that after the sample is illuminated by the structured light, the defocusing information becomes fuzzy and the information on the focal plane is clear. In the third prior art, super-resolution imaging of a sample is realized through structural light modulation of multiple modes. A variable-spacing spatial filter is utilized to realize a plurality of microscopic imaging modes in a set of system, and fluorescent polarization microscopic imaging modes are added in a fused manner on the basis. However, the above-mentioned technique can only realize structural light illumination tomography or structural light illumination super-resolution imaging, and cannot simultaneously realize detection of fluorescence anisotropy.
Disclosure of Invention
The invention aims to provide a fluorescence anisotropy detection method and a fluorescence anisotropy detection system, which are used for solving the problems in the prior art and realizing detection of fluorescence anisotropy.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a fluorescence anisotropy detection method, which comprises the following steps:
step one, polarized laser enters a stripe modulator, light rays with different diffraction orders are reflected through the stripe modulator, the light rays with different diffraction orders are filtered through a variable-pitch spatial filter, the polarization direction is adjusted, and the polarization modulation of the structural light is completed;
step two, the modulated light rays with different diffraction orders transmit excitation light to an objective lens through a dichroic mirror, and then the excitation light is conjugated to the sample surface after the objective lens to interfere, so that a structural light illumination light field is formed, meanwhile, fluorescent signals emitted by the surface of the sample illuminated by the structural light illumination light field are acquired by the objective lens, and the fluorescent signals are reflected by the dichroic mirror and generate two polarized fluorescent signals with orthogonal directions after passing through a Wollaston polarizing prism; two beams of orthogonal polarized fluorescence signals are adjusted to a camera imaging photosensitive unit;
modulating a first pattern by using a stripe modulator, modulating structure light in a vertical direction by using polarization in the first step, exciting the surface of a sample by using vertical polarization in the second step to generate a fluorescent signal, and collecting and observing the vertical fluorescent intensity and the horizontal fluorescent intensity of the fluorescent signal; modulating a second pattern by using a stripe modulator, repeating the first step of polarizing and modulating the structure light in the horizontal direction, repeating the second step of polarizing and exciting the surface of the sample by using the horizontal polarization to generate a fluorescent signal, and collecting and observing the vertical fluorescent intensity and the horizontal fluorescent intensity of the fluorescent signal; and combining the fluorescence intensity data measured by the first pattern and the second pattern to obtain the fluorescence anisotropy parameter of the sample.
Optionally, in the first step, the polarized laser is emitted by a polarized laser emitter, wherein the light beam is focused by a first focusing lens of the beam expanding lens group, the light beam is collimated and expanded by a second focusing lens after being emitted, and the polarized laser after being collimated and expanded is incident to the fringe modulator after passing through a polarized beam splitter and a half-wave plate.
Optionally, in the first step, three groups of diffracted light of different orders are reflected by the fringe modulator; the three groups of diffracted light rays pass through the half wave plate to the polarization beam splitter again, so that the light intensity ratio of + -1 level light and zero level light in the diffracted light rays is increased; after passing through the polarization beam splitter, the diffracted light is focused on the variable-pitch spatial filter through the lens; the space-variable filter keeps + -1 level light after filtering, and the polarization direction of + -1 level diffraction light is adjusted by the zoned half-wave plate, so that the polarization direction of a pair of + -1 level light is orthogonal to the connection direction of diffraction light spots of + -1 level light, and further the polarization modulation of structural light is completed.
Optionally, in the second step, the fluorescent signal is filtered through an emission filter after being reflected by a dichroic mirror, and the filtered fluorescent signal passes through a tube lens, then passes through a wollaston polarizing prism, and then passes through the wollaston polarizing prism to generate two polarized fluorescent signals with orthogonal directions; and then the polarized fluorescent signals which are divided into two beams in an orthogonal mode are adjusted to the camera imaging photosensitive unit by utilizing the beam adjusting mirror group, and the polarized fluorescent signals in two orthogonal directions are collected.
Optionally, in step three, the first pattern I is modulated v When the vertical fluorescence intensity I is observed vv Horizontal fluorescence intensity I vh ；
The fluorescence polarization parameters are expressed as:
the fluorescence radiation has one vertically polarized excitation and two identical horizontal components, so the total fluorescence intensity is expressed as:
I t ＝I vv +2I vh
fluorescence anisotropy is expressed as follows:
introducing a parameter lambda v ＝I vv /I vh The fluorescence anisotropy measured in the vertical direction is expressed as:
modulating the second pattern I h When the vertical fluorescence intensity I is observed hv Horizontal fluorescence intensity I h h, based on the above formula, the fluorescence anisotropy in the horizontal direction is expressed as:
the calculated parameter r is the result of detecting the fluorescence anisotropy of the obtained sample.
The invention also provides a fluorescence anisotropy detection system, which comprises a polarized laser transmitter, wherein the polarized laser transmitter is used for transmitting polarized laser, one end of the polarized laser transmitter is coaxially provided with a collimation and beam expansion lens group, the tail end of the collimation and beam expansion lens group is provided with a polarized beam splitter, and the polarized beam splitter is used for conducting the beam expansion light to a stripe modulator and conducting the light of different diffraction orders reflected by the stripe modulator to a variable-spacing spatial filter; the polarization beam splitter is characterized in that one end of the polarization beam splitter is provided with a first half-wave plate and a stripe modulator which are coaxially arranged, the other end of the polarization beam splitter is provided with a lens, the tail end of the lens is provided with a variable-pitch spatial filter, a partition half-wave plate and a relay lens which are coaxially arranged, polarized laser is emitted by a polarization laser emitter, light beams are focused through a first focusing lens of a beam expanding lens group, and are emitted to a second focusing lens, and the second focusing lens collimates and expands the light beams. The beam-expanded laser passes through the polarizing beam splitter and the first half wave plate to the stripe modulator, and after the laser is reflected by the stripe modulator, the beam contains three groups of diffraction light rays with different orders. The three groups of diffracted light rays pass through the first half wave plate to the polarization beam splitter again, the first half wave plate is used for adjusting the polarization direction of the light beams, and the light beams reflected by the fringe modulator pass through the first half wave plate twice to adjust the polarization direction. In addition, the + -1 level light after passing through the variable-pitch spatial filter adjusts the polarization direction by using a zoned half-wave plate. The polarization direction is adjusted by adjusting the fast axis direction of the first half wave plate, so that the light intensity ratio of + -1 level light and zero level light in diffracted light is increased, and after the light beam passes through the polarization beam splitter, the light beam can be focused on the variable-pitch spatial filter by utilizing the lens. The variable-pitch spatial filter only retains + -1 st order diffracted light, and filters out the remaining orders of diffracted light. The polarization direction of the + -1-order diffraction light is adjusted by using the zoned half-wave plate, so that the polarization direction of a pair of + -1-order light is orthogonal to the connection direction of diffraction light spots of + -1-order light, and further the polarization modulation of the structural light is completed; the end of the relay lens is sequentially provided with a dichroic mirror, an objective lens and a sample; an emission filter is arranged on one side of the dichroic mirror, the axis of the emission filter is perpendicular to the axis of the objective lens, and a second half wave plate, a tube lens and a Wollaston polarizing prism are coaxially arranged at the tail end of the emission filter; the end of the Wollaston polarizing prism is provided with a beam adjusting mirror group, and the end of the beam adjusting mirror group is provided with a camera; the + -1-order diffraction light modulated by the structured light polarization modulation module passes through the relay lens, and the dichroic mirror transmits the excitation light to the objective lens, so that + -1-order diffraction light spots on the variable-pitch spatial filter can be conjugated to the back focal plane of the objective lens. The + -1 st-order diffracted light is changed into parallel light after passing through the objective lens, and thus, is irradiated to the sample surface to interfere, thereby forming a structured light illumination field. Meanwhile, the fluorescent signals emitted by the sample surface irradiated by the structural light illumination light field are acquired by the objective lens, and the fluorescent signals are reflected by the dichroic mirror and then pass through the emission filter, so that the scattered light and the emitted light of other wave bands can be filtered. The filtered fluorescent signals pass through a tube lens and then pass through a Wollaston polarizing prism, and two polarized fluorescent signals with orthogonal directions are generated after passing through the Wollaston polarizing prism. And then the polarized fluorescent signals which are divided into two beams in an orthogonal mode are adjusted to the imaging photosensitive unit of the camera by the beam adjusting mirror group, namely, the camera can collect fluorescent polarized signals in two orthogonal directions at the same time.
Optionally, the stripe modulator is loaded with equidistant black and white stripes, which can generate spatial modulation on the amplitude of light and generate light beams with different diffraction orders; the fringe modulator may be a liquid crystal spatial light modulator, a digital micromirror array or a mechanical grating. As a wavefront modulation device, a fringe modulator is used as a diffraction grating in the present system. By loading equally spaced black and white stripes on the stripe modulator, spatial modulation of the light amplitude is produced. When polarized light is projected to the fringe modulator, light beams with different diffraction orders are generated under the grating action generated by black and white fringes.
Optionally, the variable-pitch spatial filter includes an opaque substrate, and two light-passing holes are formed on the substrate; the light through hole is positioned at the position of + -1-level light of the structured light illumination confocal microscopic imaging; the relay lens consists of two lenses, and the relay lens is arranged at the rear end of the partition half-wave plate. The two lenses are used for conjugating the diffracted light to a focusing plane behind the objective lens, and the two parallel diffracted light beams are still parallel after passing through the objective lens.
Optionally, a dichroic mirror is located at the rear end of the relay lens in the present system, the parallel light beam is transmitted to the objective lens through the dichroic mirror, interference is formed at the focusing surface, and then the excited fluorescence is reflected to the emission filter through the dichroic mirror. The dichroic mirror is of two types, short-wavelength and long-wavelength, wherein the short-wavelength dichroic mirror is capable of reflecting light of a set short-wavelength range and transmitting light of a set long-wavelength range. The long-wave dichroic mirror is capable of reflecting light in a set long-wavelength range and transmitting light in a set short-wavelength range; the Wollaston polarizing prism can generate two linearly polarized lights which are mutually separated and have mutually perpendicular vibration directions by the incident fluorescent signals, and excited fluorescence reaches the Wollaston polarizing prism through the emission filter and the tube lens and is then decomposed into two polarized lights with mutually perpendicular polarization directions; the light beam adjusting mirror group comprises four plane mirrors; the two linearly polarized light beams can be reflected to the photosensitive unit of the camera at any angle.
Compared with the prior art, the invention has the following technical effects:
according to the fluorescence anisotropy detection method, the stripe modulator is used for generating the multi-order diffraction light beam, and the variable-spacing spatial filter is used for obtaining the + -1-order diffraction light beam which can interfere. The invention relates to a fluorescence detection module, which utilizes a polarization beam splitter to split light and simultaneously detects polarized fluorescence images in two orthogonal directions on a camera target surface. Since the polarization direction of the illumination stripe is consistent with the stripe direction, the vertical fluorescence intensity I can be detected simultaneously on the camera after polarization light splitting vv And horizontal fluorescence intensity I vh . Further, in order to obtain super-resolution or tomographic signals in another direction during the structured light imaging, the illumination stripes may be rotated 90 degrees, so that the vertical fluorescence intensity I can be detected simultaneously without rotating the polarization detection hv And horizontal fluorescence intensity I hh For calculating r values in two directions corresponding to each pixel and obtaining a structured light super-resolution or structured light tomography result. Meanwhile, the pixel area with fluorescent molecules can be selected by utilizing structured light super-resolution or structured light chromatography, so that the estimated deviation of fluorescence anisotropy caused by the fact that the detected light intensity is 0 or the detected light intensity is the background on a background pixel is avoided.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the arrangement of a fluorescence anisotropy detection system of the present invention;
FIG. 2 is a schematic diagram of a polarized fluorescence excitation pattern generated by a vertical stripe modulator according to the present invention;
FIG. 3 is a schematic diagram of a polarized fluorescence CCD camera acquisition pattern generated by a stripe modulator in the vertical direction of the present invention;
FIG. 4 is a schematic diagram of a polarized fluorescence excitation pattern generated by a horizontal stripe modulator of the present invention;
FIG. 5 is a schematic diagram of a polarized fluorescence CCD camera acquisition pattern generated by a stripe modulator in the horizontal direction according to the present invention;
reference numerals illustrate: the device comprises a 1-polarized laser transmitter, a 2-collimation beam expansion lens group, a 3-polarized beam splitter, a 4-first half-wave plate, a 5-stripe modulator, a 6-lens, a 7-variable space filter, an 8-partition half-wave plate, a 9-relay lens, a 10-objective lens, a 11-dichroic mirror, a 12-emission filter, a 13-second half-wave plate, a 14-tube lens, a 15-Wollaston polarizing prism, a 16-beam adjusting mirror group, a 17-CCD camera, a 18-sample and a 19-reflecting mirror.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention aims to provide a fluorescence anisotropy detection method and a fluorescence anisotropy detection system, which are used for solving the problems in the prior art and realizing detection of fluorescence anisotropy.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.
As shown in fig. 1, the invention provides a fluorescence anisotropy detection system, which comprises a polarized laser transmitter 1, wherein the polarized laser transmitter 1 is used for transmitting polarized laser, one end of the polarized laser transmitter 1 is coaxially provided with a collimation and beam expansion lens group 2, the tail end of the collimation and beam expansion lens group 2 is provided with a reflecting mirror 19, and the emergent end of the reflecting mirror 19 is provided with a polarized beam splitter 3; one end of the polarization beam splitter 3 is provided with a first half-wave plate 4 and a stripe modulator 5 which are coaxially arranged, the other end of the polarization beam splitter is provided with a lens 6, the tail end of the lens 6 is provided with two symmetrically arranged reflecting mirrors 19, and the tail end of the symmetrically arranged reflecting mirrors 19 is provided with a variable-pitch spatial filter 7, a partition half-wave plate 8 and a relay lens 9 which are coaxially arranged; the end of the relay lens 9 is provided with a dichroic mirror 11, an objective lens 10 and a sample 18 in sequence; an emission filter 12 is arranged on one side of the dichroic mirror 11, the axis of the emission filter 12 is perpendicular to the axis of the objective lens 10, and a second half-wave plate 13, a tube lens 14 and a Wollaston polarizing prism 15 are coaxially arranged at the tail end of the emission filter 12; the tail end of the Wollaston polarizing prism 15 is provided with a light beam adjusting mirror group 16, the tail end of the light beam adjusting mirror group 16 is provided with a CCD camera 17, and the light beam adjusting mirror group 16 comprises four plane mirrors; a photosensitive unit capable of reflecting two linearly polarized light beams to the CCD camera 17 at an arbitrary angle; the CCD camera 17 refers to an imaging detection device composed of two-dimensional pixels, and can also be of other types such as CMOS, sCMOS and the like, without changing the essence of the invention.
As shown in fig. 2, 3, 4 and 5, the polarized fluorescence excitation patterns generated by the stripe modulators in the vertical direction and the horizontal direction are collected by the corresponding CCD cameras 17.
Wherein the stripe modulator 5 in the vertical direction modulates the pattern as I v . In the acquired image, the vertical fluorescence intensity I of the fluorescent sample is observed vv Horizontal fluorescence intensity I of the fluorescent sample was observed vh . The stripe modulator 5 in the horizontal direction modulates the pattern as I h . In the acquired image, the vertical fluorescence intensity I of the fluorescent sample is observed hv Horizontal fluorescence intensity I of the fluorescent sample was observed hh 。
The invention provides a fluorescence anisotropy detection method based on structured light illumination, which comprises the following steps:
step one, structured light polarization modulation
The polarized laser is emitted by the polarized laser emitter 1, passes through the collimating and beam expanding lens group 2, wherein the light beam is focused by the first focusing lens of the collimating and beam expanding lens group 2, and is emitted to the second focusing lens, and the second focusing lens collimates and expands the light beam. The expanded laser light passes through the polarizing beam splitter 3 and the first half-wave plate 4 to the fringe modulator 5. The stripe modulator 5 is used as a diffraction grating by loading uniformly spaced black and white stripes on the stripe modulator 5 and changing the positions of the black and white stripes. The beam-expanded laser can obtain the diffraction fringes of the optical field of the structured light with different phases through the fringe modulator 5. In the system, the light beam reflected by the fringe modulator 5 contains light rays with different diffraction orders, passes through the first half-wave plate 4 to the polarization beam splitter 3 again, and adjusts the polarization direction by adjusting the fast axis direction of the first half-wave plate 4, so that the light intensity ratio of + -1-order light and zero-order light in the diffracted light is increased. After passing through the polarizing beam splitter 3, the light beam can be focused on a variable pitch spatial filter 7 by means of a lens 6. The variable pitch spatial filter 7 retains only ±1 order diffracted light, and filters out the remaining orders of diffracted light. The polarization direction of the + -1-order diffraction light is adjusted by the zonal half-wave plate 8, so that the polarization direction of a pair of + -1-order light is orthogonal to the connection line direction of diffraction light spots of + -1-order light, and further the polarization modulation of the structural light is completed.
Step two, polarized fluorescence signal perception
The modulated ±1-order diffracted light passes through the relay lens 9, and the ±1-order diffracted light transmits excitation light to the objective lens 10 through the dichroic mirror 11, whereby the ±1-order diffracted light spot on the variable pitch spatial filter 7 can be conjugated to the back focal plane of the objective lens 10. The ±1-order diffracted light becomes parallel light after passing through the objective lens 10, and thus, is irradiated to the surface of the sample 18 to interfere, thereby forming a structured light illumination field. Meanwhile, the fluorescent signal emitted by the surface of the sample 18 irradiated by the structured light illumination field is acquired by the objective lens, and the fluorescent signal is reflected by the dichroic mirror 11 and then passes through the emission filter 12, so that the scattered light and the emitted light of other wave bands can be filtered. The filtered fluorescence signal passes through the tube lens 14 and then passes through the Wollaston polarizing prism 15, and two polarized fluorescence signals with orthogonal directions are generated after passing through the Wollaston polarizing prism 15. And the polarized fluorescence signals which are divided into two beams in an orthogonal mode are adjusted to an imaging photosensitive unit of the CCD camera 17 by using the beam adjusting mirror group 16, namely the CCD camera 17 can collect the polarized fluorescence signals in two orthogonal directions at the same time.
Step three, fluorescence anisotropy detection
First modulating pattern I with a stripe modulator 5 v Generating vertically polarized structured light, exciting a fluorescent sample using vertically polarization, capturing an image by the CCD camera 17, and then observing the vertical fluorescence intensity I of the fluorescent sample vv Horizontal fluorescence intensity I of the fluorescent sample was observed vh . Modulating pattern I with a fringe modulator 5 h Producing horizontally polarized structured light. The fluorescence sample is excited using horizontal polarization, an image is acquired by the CCD camera 17, and then the vertical fluorescence intensity I of the fluorescence sample is observed hv Horizontal fluorescence intensity I of the fluorescent sample was observed hh . Further, fluorescence anisotropy parameter r was obtained.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "top", "bottom", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The principles and embodiments of the present invention have been described in detail with reference to specific examples, which are provided to facilitate understanding of the method and core ideas of the present invention; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.
Claims (9)
1. A fluorescence anisotropy detection method is characterized in that: the method comprises the following steps:
step one, polarized laser enters a stripe modulator, light rays with different diffraction orders are reflected through the stripe modulator, the light rays with different diffraction orders are filtered through a variable-pitch spatial filter, the polarization direction is adjusted, and the polarization modulation of the structural light is completed;
step two, the modulated light rays with different diffraction orders transmit excitation light to an objective lens through a dichroic mirror, and then the excitation light is conjugated to the sample surface after the objective lens to interfere, so that a structural light illumination light field is formed, meanwhile, fluorescent signals emitted by the surface of the sample illuminated by the structural light illumination light field are acquired by the objective lens, and the fluorescent signals are reflected by the dichroic mirror and generate two polarized fluorescent signals with orthogonal directions after passing through a Wollaston polarizing prism; two beams of orthogonal polarized fluorescence signals are adjusted to a camera imaging photosensitive unit;
modulating a first pattern by using a stripe modulator, modulating structure light in a vertical direction by using polarization in the first step, exciting the surface of a sample by using vertical polarization in the second step to generate a fluorescent signal, and collecting and observing the vertical fluorescent intensity and the horizontal fluorescent intensity of the fluorescent signal; modulating a second pattern by using a stripe modulator, repeating the first step of polarizing and modulating the structure light in the horizontal direction, repeating the second step of polarizing and exciting the surface of the sample by using the horizontal polarization to generate a fluorescent signal, and collecting and observing the vertical fluorescent intensity and the horizontal fluorescent intensity of the fluorescent signal; and combining the fluorescence intensity data measured by the first pattern and the second pattern to obtain the fluorescence anisotropy parameter of the sample.
2. The fluorescence anisotropy detection method of claim 1, wherein: in the first step, polarized laser is emitted by a polarized laser emitter, wherein the light beam is focused by a first focusing lens of a beam expanding lens group, the light beam is collimated and expanded by a second focusing lens after being emitted, and the polarized laser after being collimated and expanded enters the fringe modulator after passing through a polarized beam splitter and a half-wave plate.
3. The fluorescence anisotropy detection method of claim 1, wherein: in the first step, three groups of diffraction light rays with different orders are reflected by a stripe modulator; the three groups of diffracted light rays pass through the half wave plate to the polarization beam splitter again, so that the light intensity ratio of + -1 level light and zero level light in the diffracted light rays is increased; after passing through the polarization beam splitter, the diffracted light is focused on the variable-pitch spatial filter through the lens; the space-variable filter keeps + -1 level light after filtering, and the polarization direction of + -1 level diffraction light is adjusted by the zoned half-wave plate, so that the polarization direction of a pair of + -1 level light is orthogonal to the connection direction of diffraction light spots of + -1 level light, and further the polarization modulation of structural light is completed.
4. The fluorescence anisotropy detection method of claim 1, wherein: in the second step, the fluorescent signal is filtered through an emission filter after being reflected by a dichroic mirror, and the filtered fluorescent signal passes through a Wollaston polarizing prism after passing through a tube lens, and then two polarized fluorescent signals with orthogonal directions are generated after passing through the Wollaston polarizing prism; and then the polarized fluorescent signals which are divided into two beams in an orthogonal mode are adjusted to the camera imaging photosensitive unit by utilizing the beam adjusting mirror group, and the polarized fluorescent signals in two orthogonal directions are collected.
5. The fluorescence anisotropy detection method of claim 1, wherein: in step three, the first pattern I is modulated v When the vertical fluorescence intensity I is observed vv Horizontal fluorescence intensity I vh ；
The fluorescence polarization parameters are expressed as:
the fluorescence radiation has one vertically polarized excitation and two identical horizontal components, so the total fluorescence intensity is expressed as:
I t ＝I vv +2I vh
fluorescence anisotropy is expressed as follows:
introducing a ginsengThe number lambda v ＝I vv /I vh The fluorescence anisotropy measured in the vertical direction is expressed as:
modulating the second pattern I h When the vertical fluorescence intensity I is observed hv Horizontal fluorescence intensity I h h, based on the above formula, the fluorescence anisotropy in the horizontal direction is expressed as:
the calculated parameter r is the result of detecting the fluorescence anisotropy of the obtained sample.
6. A fluorescence anisotropy detection system, characterized by: the device comprises a polarized laser transmitter, a collimating and beam expanding lens group and a polarization beam splitter, wherein the polarized laser transmitter is used for transmitting polarized laser, one end of the polarized laser transmitter is coaxially provided with the collimating and beam expanding lens group, and the tail end of the collimating and beam expanding lens group is provided with the polarization beam splitter; one end of the polarization beam splitter is provided with a first half-wave plate and a stripe modulator which are coaxially arranged, the other end of the polarization beam splitter is provided with a lens, and the tail end of the lens is provided with a variable-pitch spatial filter, a partition half-wave plate and a relay lens which are coaxially arranged; the end of the relay lens is sequentially provided with a dichroic mirror, an objective lens and a sample; an emission filter is arranged on one side of the dichroic mirror, the axis of the emission filter is perpendicular to the axis of the objective lens, and a second half wave plate, a tube lens and a Wollaston polarizing prism are coaxially arranged at the tail end of the emission filter; the end of the Wollaston polarizing prism is provided with a light beam adjusting mirror group, and the end of the light beam adjusting mirror group is provided with a camera.
7. The fluorescence anisotropy detection system of claim 6, wherein: the fringe modulator is loaded with equidistant black and white fringes, can generate spatial modulation on the amplitude of light, and generate light beams with different diffraction orders.
8. The fluorescence anisotropy detection system of claim 6, wherein: the variable-pitch spatial filter comprises an opaque substrate, wherein two light transmission holes are formed in the substrate; the light passing hole is positioned at the position of + -1-level light of the structured light illumination confocal microscopic imaging.
9. The fluorescence anisotropy detection system of claim 6, wherein: the Wollaston polarizing prism can generate two linearly polarized lights which are mutually separated and have mutually perpendicular vibration directions from an incident fluorescent signal; the light beam adjusting mirror group comprises four plane mirrors; the two linearly polarized light beams can be reflected to the photosensitive unit of the camera at any angle.
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