CN115015200B - Nanometer precision fluorescence imaging device and method based on spatial light modulation - Google Patents
Nanometer precision fluorescence imaging device and method based on spatial light modulation Download PDFInfo
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Abstract
The invention provides a nanometer precision fluorescent imaging device based on spatial light modulation, which is characterized by comprising an excitation module, an activation module and an imaging module, wherein the excitation module and the activation module comprise single micro-element adjustable spatial light modulators, excitation light rays which are shaped and emitted by the excitation module are combined with activation light rays which are shaped and emitted by the activation module to obtain combined light rays, and the combined light rays are amplified and irradiated onto a sample in nanometer size and imaged by the imaging module; the device provided by the invention realizes novel laser spot modulation without mechanical movement and laser scanning, can distinguish single fluorescent molecules or subcellular fine structures, greatly reduces the complexity of a fluorescent imaging device, and improves the positioning and imaging efficiency.
Description
Technical Field
The invention relates to the field of fluorescence imaging, in particular to a nanometer precision fluorescence imaging method based on spatial light modulation.
Background
Biotechnology and fluorescence imaging technology are the most rapidly developing and popular scientific fields in the 21 st century. Although STED, MINFLUX and other fluorescence imaging methods successively overcome the limit of diffraction limit and reach the transverse resolution capability of 100nm or even 10nm, the method is difficult to actively and positively position biomolecules, subcellular fine structures and the like accurately. The STED fluorescence microscopy technology utilizes stimulated radiation to selectively consume excited fluorescent molecules in an edge area in an excitation light spot so as to reduce the luminous range of effective fluorescence, compress the effective PSF (point spread function) scale and improve the resolution of a system. But there are also corresponding disadvantages such as: 1. the loss light needs strong brightness, and the cost of the laser is high; 2. the damage to the sample is larger; 3. synchronous positioning of the excitation light spot and the loss light spot is complex, and operation is quite complicated; 4. the stability requirement on the system is very high, and frequent inspection and system maintenance are required; 5. the resolution is limited by factors such as the quality of the loss light spot, bleaching of the fluorescent sample, error in light path calibration and the like; 6. it is difficult to actively and positively precisely locate biomolecules, subcellular fine structures, and the like. By combining the MINFLUX fluorescence microscopy techniques (super-resolution imaging is realized by adding a doughnut-shaped loss light to the periphery of the traditional excitation light, so that the actually excited region is greatly reduced) and single-molecule positioning microscopy (super-resolution is realized by randomly lighting fluorescent molecules and then performing Gaussian fitting positioning), the effect that fluorescent molecules can be randomly lighted in a small region, and then the doughnut-shaped laser is used as excitation and then positioning is realized. The same MINFLUX suffers from some drawbacks such as: 1. the manufacturing cost is high; 2. the light path is complex, and the operation is quite complicated; 3. the stability of the system is high, and frequent inspection and system maintenance are required.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a nanometer precision fluorescent imaging device based on spatial light modulation, which realizes novel laser spot modulation without mechanical motion and laser scanning, can distinguish single fluorescent molecules or subcellular fine structures, greatly reduces the complexity of the fluorescent imaging device, and improves the positioning and imaging efficiency.
The invention adopts the following technical scheme:
The nanometer precision fluorescent imaging device based on the spatial light modulation comprises an excitation module, an activation module and an imaging module, wherein the excitation module and the activation module comprise single infinitesimal adjustable spatial light modulators, excitation light rays emitted by the excitation module in a shaping mode are combined with activation light rays emitted by the activation module in a shaping mode to obtain combined light rays, and the combined light rays are amplified and irradiated onto a sample in nanometer size and imaged through the imaging module.
Specifically, the excitation module sequentially comprises, according to the propagation direction of the optical path: a laser light source I, a beam expander I, a uniform light lens group I, a focusing lens I, a total internal reflection lens I and a spatial light modulator I; the beam expander I is used for expanding the excitation light rays emitted by the laser light source I; the uniform light lens group I and the focusing lens I are used for converging the exciting light rays; the total internal reflection lens I makes the exciting light ray enter the spatial light modulator I at a specific angle; the spatial light modulator I shapes and emits the incident excitation light.
Specifically, the activation module sequentially includes, according to a propagation direction of an optical path: the device comprises a laser light source II, a beam expander II, a uniform light lens group II, a focusing lens II, a total internal reflection lens II, a spatial light modulator II, a dichroic mirror, a collimating lens group, a filter lens turntable, an objective lens and an objective table; the beam expander II is used for expanding the activated light rays emitted by the laser light source II; the uniform light lens group II and the focusing lens II are used for converging the activated light, the activated light of the total internal reflection lens II is incident into the spatial light modulator II at a specific angle, the activated light is shaped by the spatial light modulator II and then is incident onto the dichroic mirror at a specific angle, and combined light is formed with the exciting light; the optical filter turntable reflects the combined light to the objective lens; the combined light is amplified by the collimator lens group and the objective lens and then irradiated on the sample in nanometer size.
Specifically, the imaging module sequentially comprises, according to the propagation direction of the optical path: a piezoelectric ceramic motor, a tube mirror and a detector; the piezoelectric ceramic motor realizes axial precise focusing; the fluorescence is transmitted through the optical filter turntable and enters the detector after being focused by the tube mirror.
In another aspect, the embodiment of the invention provides a nanometer precision fluorescence imaging method based on spatial light modulation, which comprises the following steps:
1) Firstly, placing a sample on an objective table, and controlling all micro-mirrors of a spatial light modulator I to be on by an upper computer; then, the position of the objective lens relative to the objective table is roughly adjusted, so that the imaging of the detector reaches better definition; then the position of the objective lens relative to the objective table is finely adjusted by the piezoelectric ceramic motor, so that the imaging of the detector reaches the optimal definition;
2) According to the position information and the shape information of the ROI area, controlling a spatial light modulator I to shape the exciting light into a geometric shape I; controlling a spatial light modulator II to reshape the activated light into a geometric shape II; wherein the locations of geometry I and geometry II may be inclusive, intersecting, or mutually exclusive;
3) The fluorescent positioning algorithm and the single infinitesimal adjustable spatial light modulator I and the spatial light modulator II are used for realizing the real-time adjustment of the central positions of the geometric shape I and the geometric shape II; recording fluorescent molecules of the ROI area by continuously iterating the central position;
4) Obtaining the positions of fluorescent molecules in the region of the ROI recorded by the central positions of the geometric shapes I and II from the step 3); and fluorescence emitted by the fluorescent molecules is transmitted through the optical filter turntable, focused by the tube mirror, and then acquired by the detector for imaging.
In particular, the spatial light modulators I, II are single micro-element tunable spatial light modulators, including but not limited to DMDs or transmissive LCDs.
Specifically, geometry II is contained in geometry I at the iteration center position; geometry ii and geometry i include, but are not limited to, doughnut-shaped, rectangular or triangular, and the like.
Specifically, in the positioning process of the fluorescence positioning algorithm, the spatial light modulators I and II are controlled to form light spots and project the light spots into a sample area, then the spatial light modulators I and II are adjusted, the micro-mirrors at different positions are switched on to enable the light spots to move on the circumference with randomly started positions as circle centers and L=600nm as diameters to complete one iteration, the diameter L is reduced after the iteration is completed to conduct the next iteration, the operation is repeated continuously, the iteration is ended until the diameter L=60 nm, the position of a fluorescent molecule is finally determined, and the distance r between the fluorescent molecule and the nearest light spot center point in the iteration process can be expressed as:
Wherein, The representation is an estimate, the MLE representation is a maximum likelihood method, the maximum likelihood estimate/>The result of (a) is approximately expressed as polynomial distribution/>, of the probability of fluorescent molecules being closest to the spot center point in an iterationThe following formula is shown:
Wherein the method comprises the steps of Is a collection of photon numbers collected by the detector during the movement of the doughnut light spot; n is the total number of photons collected during a single iteration; n 0!…nk-1 -! Is/>The elements in the set represent the number of photons collected each time geometry I, II is moved in a single iteration; k is the number of times the central position of the geometric shapes I and II is moved by the control geometric shapes I and II of the spatial light modulators I and II in the single iteration process; p i is a vector parameter,/>Where lambda i denotes the wavelength of the background noise caused by geometry i and lambda bi denotes the wavelength of the background noise caused by geometry ii.
As can be seen from the above description of the present invention, compared with the prior art, the present invention has the following advantages:
(1) The method provides a nanometer precision fluorescent imaging device based on spatial light modulation, which comprises an excitation module, an activation module and an imaging module, wherein the excitation module and the activation module comprise single micro-element adjustable spatial light modulators, excitation light rays shaped and emitted by the excitation module are combined with activation light rays shaped and emitted by the activation module to obtain combined light rays, the combined light rays are amplified and irradiated on a sample in nanometer size and imaged through the imaging module, and novel laser spot modulation without mechanical movement and laser scanning is realized; and the realized nanometer precision fluorescence microscopic imaging can distinguish single fluorescent molecules or subcellular fine structures.
(2) The novel laser doughnut light spot modulation method provided by the method adopts the MEMS device as a spatial light modulator, so that the complexity of the fluorescence imaging device is greatly reduced.
(3) The invention uses a single infinitesimal adjustable spatial light modulator and combines a fluorescent molecule positioning algorithm to realize active and active fluorescent molecule positioning and imaging, and improves the positioning and imaging efficiency.
Drawings
FIG. 1 shows a nanometer-precision fluorescence imaging device based on spatial light modulation according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a two-dimensional grating diffraction solution according to an embodiment of the present invention; wherein (a) diffraction phenomenon, (b) expansion of diffraction interval, (c) retention of zero-order diffraction.
The invention is further described in detail below with reference to the drawings and the specific examples.
Detailed Description
The invention provides a nanometer precision fluorescent imaging device based on spatial light modulation, which realizes novel laser spot modulation without mechanical motion and laser scanning, can distinguish single fluorescent molecules or subcellular fine structures, greatly reduces the complexity of the fluorescent imaging device and improves the positioning and imaging efficiency.
Referring to fig. 1, a spatial light modulation-based nano-precision fluorescence imaging device provided by an embodiment of the present invention includes an excitation module, an activation module, and an imaging module, where the excitation module and the activation module include a single micro-element adjustable spatial light modulator, and excitation light rays emitted from the excitation module after shaping are combined with activation light rays emitted from the activation module after shaping, so as to obtain combined light rays, and the combined light rays are amplified, irradiated onto a sample in nano-size, and imaged by the imaging module.
Specifically, the excitation module sequentially comprises, according to the propagation direction of the optical path: a laser light source I-1, a beam expander I-2, a uniform light lens group I-3, a focusing lens I-4, a total internal reflection lens I-5 and a spatial light modulator I-6; the beam expander I-2 is used for expanding the excitation light rays emitted by the laser light source I-1; the uniform light lens group I-3 and the focusing lens I-4 are used for converging the exciting light rays; the total internal reflection lens I-5 makes the exciting light ray incident into the spatial light modulator I-6 at a specific angle; the spatial light modulator I-6 shapes and emits the incident excitation light.
Specifically, the activation module sequentially includes, according to a propagation direction of an optical path: the device comprises a laser light source II-7, a beam expander II-8, a uniform light lens group II-9, a focusing lens II-10, a total internal reflection lens II-11, a spatial light modulator II-12, a dichroic mirror-13, a collimating lens group-14, a filter turntable-15, an objective lens-17 and an objective table-16; the beam expander II-8 is used for expanding the activating light rays emitted by the laser light source II-7; the uniform light lens group II-9 and the focusing lens II-10 are used for converging the activated light, the activated light of the total internal reflection lens II-11 is incident into the spatial light modulator II-12 at a specific angle, and the activated light is shaped by the spatial light modulator II-12 and then is incident onto the dichroic mirror-13 at a specific angle to form combined light with the exciting light; the optical filter turntable 15 reflects the combined light to the objective lens-17; the combined light is amplified by the collimator lens assembly-14 and the objective lens-17 and then irradiated onto the sample on the stage-16 in nanometer size.
Specifically, the imaging module sequentially comprises, according to the propagation direction of the optical path: the piezoelectric ceramic motor-18, the tube mirror-19 and the detector-20; the piezoelectric ceramic motor-18 realizes axial precise focusing; the fluorescence is transmitted through the filter turret-15 and focused by the tube mirror-19 before entering the detector-20.
According to the embodiment of the invention, the two-dimensional grating diffraction problem occurring when the spatial light modulator modulates the nanometer precision light spot is solved through the combination of the relay lens group and the focusing lens; the principle is that a relay convex lens in an optical path is utilized to amplify laser spots, the interval between diffraction stages is simultaneously amplified, then only zero-order diffraction spots are reserved to enter the optical path, and then the zero-order spots are compressed to the initial size by a focusing lens; fig. 2 is a schematic diagram in which (a) diffraction phenomenon, (b) diffraction interval is enlarged, (c) zero-order diffraction is reserved.
In another aspect, the embodiment of the invention provides a nanometer precision fluorescence imaging method based on spatial light modulation, which comprises the following steps:
1) Firstly, placing a sample on an objective table, and controlling all micro-mirrors of a spatial light modulator I to be on by an upper computer; then, the position of the objective lens relative to the objective table is roughly adjusted, so that the imaging of the detector reaches better definition; then the position of the objective lens relative to the objective table is finely adjusted by the piezoelectric ceramic motor, so that the imaging of the detector reaches the optimal definition;
2) According to the position information and the shape information of the ROI area, controlling a spatial light modulator I to shape the exciting light into a geometric shape I; controlling a spatial light modulator II to reshape the activated light into a geometric shape II; wherein the locations of geometry I and geometry II may be inclusive, intersecting, or mutually exclusive;
3) The fluorescent positioning algorithm and the single infinitesimal adjustable spatial light modulator I and the spatial light modulator II are used for realizing the real-time adjustment of the central positions of the geometric shape I and the geometric shape II; recording fluorescent molecules of the ROI area by continuously iterating the central position;
4) Obtaining the positions of fluorescent molecules in the region of the ROI recorded by the central positions of the geometric shapes I and II from the step 3); and fluorescence emitted by the fluorescent molecules is transmitted through the optical filter turntable, focused by the tube mirror, and then acquired by the detector for imaging.
In particular, the spatial light modulators I, II are single micro-element tunable spatial light modulators, including but not limited to DMDs or transmissive LCDs.
Specifically, geometry II is contained in geometry I at the iteration center position; geometry ii and geometry i include, but are not limited to, doughnut-shaped, rectangular or triangular, and the like.
Specifically, in the positioning process of the fluorescence positioning algorithm, the spatial light modulators I and II are controlled to form light spots and project the light spots into a sample area, then the spatial light modulators I and II are adjusted, the micro-mirrors at different positions are switched on to enable the light spots to move on the circumference with randomly started positions as circle centers and L=600nm as diameters to complete one iteration, the diameter L is reduced after the iteration is completed to conduct the next iteration, the operation is repeated continuously, the iteration is ended until the diameter L=60 nm, the position of a fluorescent molecule is finally determined, and the distance r between the fluorescent molecule and the nearest light spot center point in the iteration process can be expressed as:
Wherein, The representation is an estimate, the MLE representation is a maximum likelihood method, the maximum likelihood estimate/>The result of (a) is approximately expressed as polynomial distribution/>, of the probability of fluorescent molecules being closest to the spot center point in an iterationThe following formula is shown:
Wherein the method comprises the steps of Is a collection of photon numbers collected by the detector during the movement of the doughnut light spot; n is the total number of photons collected during a single iteration; n 0!…nk-1 -! Is/>The elements in the set represent the number of photons collected each time geometry I, II is moved in a single iteration; k is the number of times the central position of the geometric shapes I and II is moved by the control geometric shapes I and II of the spatial light modulators I and II in the single iteration process; p i is a vector parameter,/>Where lambda i denotes the wavelength of the background noise caused by geometry i and lambda bi denotes the wavelength of the background noise caused by geometry ii. It is necessary to specify:
the excitation light source and the loss light source can be monochromatic laser light sources with other different wavelengths.
Any spatial light modulator that satisfies the above-described single-element adjustability should be considered an alternative to the method of the present invention.
The spatial light modulators I, II should be regarded as alternatives to the method of the invention as long as they follow a mutually nested manner to effect shaping and adjustment of the laser spot.
The relay lens group and the focusing lens group solve the problem of two-dimensional grating diffraction when the digital micro-mirror device modulates the nanometer precision light spot; similar effects are achieved by other measures, which should be regarded as alternatives to the method of the invention.
The detector, whatever the means for detecting fluorescence, such as a camera, is considered an alternative to the method of the present invention.
The piezoelectric ceramic motor, whatever the device for realizing axial focusing, is regarded as an alternative to the method of the invention.
The method provides a nanometer precision fluorescent imaging device based on spatial light modulation, which comprises an excitation module, an activation module and an imaging module, wherein the excitation module and the activation module comprise single micro-element adjustable spatial light modulators, excitation light rays shaped and emitted by the excitation module are combined with activation light rays shaped and emitted by the activation module to obtain combined light rays, the combined light rays are amplified and irradiated on a sample in nanometer size and imaged through the imaging module, and novel laser spot modulation without mechanical movement and laser scanning is realized; and the realized nanometer precision fluorescence microscopic imaging can distinguish single fluorescent molecules or subcellular fine structures.
The novel laser doughnut light spot modulation method provided by the method adopts the MEMS device as a spatial light modulator, so that the complexity of the fluorescence imaging device is greatly reduced.
The invention uses a single infinitesimal adjustable spatial light modulator and combines a fluorescent molecule positioning algorithm to realize active fluorescent molecule positioning and imaging, and improves the positioning and imaging efficiency.
The foregoing is merely illustrative of specific embodiments of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modification of the present invention by using the design concept shall fall within the scope of the present invention.
Claims (6)
1. The nanometer precision fluorescent imaging device based on the spatial light modulation is characterized by comprising an excitation module, an activation module and an imaging module, wherein the excitation module and the activation module comprise single micro-element adjustable spatial light modulators, excitation light rays shaped and emitted by the excitation module are combined with activation light rays shaped and emitted by the activation module to obtain combined light rays, and the combined light rays are amplified and irradiated onto a sample in nanometer size and imaged by the imaging module;
The excitation module sequentially comprises: a laser light source I, a beam expander I, a uniform light lens group I, a focusing lens I, a total internal reflection lens I and a spatial light modulator I; the beam expander I is used for expanding the excitation light rays emitted by the laser light source I; the uniform light lens group I and the focusing lens I are used for converging the exciting light rays; the total internal reflection lens I makes the exciting light ray enter the spatial light modulator I at a specific angle; the spatial light modulator I shapes and emits the incident exciting light rays;
The activation module sequentially comprises: the device comprises a laser light source II, a beam expander II, a uniform light lens group II, a focusing lens II, a total internal reflection lens II, a spatial light modulator II, a dichroic mirror, a collimating lens group, a filter lens turntable, an objective lens and an objective table; the beam expander II is used for expanding the activated light rays emitted by the laser light source II; the uniform light lens group II and the focusing lens II are used for converging the activated light, the activated light of the total internal reflection lens II is incident into the spatial light modulator II at a specific angle, the activated light is shaped by the spatial light modulator II and then is incident onto the dichroic mirror at a specific angle, and combined light is formed with the exciting light; the optical filter turntable reflects the combined light to the objective lens; the combined light is amplified by the collimating lens group and the objective lens and then irradiated on the sample on the object stage in nanometer size.
2. The spatial light modulation-based nano-precision fluorescence imaging device according to claim 1, wherein the imaging module sequentially comprises: a piezoelectric ceramic motor, a tube mirror and a detector; the piezoelectric ceramic motor realizes axial precise focusing; the fluorescence is transmitted through the optical filter turntable and enters the detector after being focused by the tube mirror.
3. A method of spatial light modulation based nano-precision fluorescence imaging using the device of any one of claims 1-2, comprising the steps of:
1) Firstly, placing a sample on an objective table, and controlling all micro-mirrors of a spatial light modulator I to be on by an upper computer; then, the position of the objective lens relative to the objective table is roughly adjusted, so that the imaging of the detector reaches better definition; then the position of the objective lens relative to the objective table is finely adjusted by the piezoelectric ceramic motor, so that the imaging of the detector reaches the optimal definition;
2) According to the position information and the shape information of the ROI area, controlling a spatial light modulator I to shape the exciting light into a geometric shape I; controlling a spatial light modulator II to reshape the activated light into a geometric shape II; wherein the locations of geometry I and geometry II may be inclusive, intersecting, or mutually exclusive;
3) The fluorescent positioning algorithm and the single infinitesimal adjustable spatial light modulator I and the spatial light modulator II are used for realizing the real-time adjustment of the central positions of the geometric shape I and the geometric shape II; recording fluorescent molecules of the ROI area by continuously iterating the central position;
4) Obtaining the positions of fluorescent molecules in the region of the ROI recorded by the central positions of the geometric shapes I and II from the step 3); and fluorescence emitted by the fluorescent molecules is transmitted through the optical filter turntable, focused by the tube mirror, and then acquired by the detector for imaging.
4. A method of nano-precision fluorescence imaging based on spatial light modulation according to claim 3, wherein the spatial light modulator i, ii is a single micro-element tunable spatial light modulator, including but not limited to DMD or transmissive LCD.
5. A method of fluorescence imaging with nanometer precision based on spatial light modulation according to claim 3, wherein geometry ii is contained in geometry i at the iteration center position; geometry ii and geometry i include, but are not limited to, doughnut-shaped, rectangular or triangular, and the like.
6. The method of claim 3, wherein the fluorescence positioning algorithm forms a light spot by controlling the spatial light modulators i and ii and projects the light spot into the sample area, then adjusts the spatial light modulators i and ii, and switches the micromirrors at different positions to move the light spot on a circumference with a randomly starting position as a center and a diameter of l=600 nm to complete an iteration, and reduces the diameter L to perform the next iteration after the iteration is completed, and the above operation is repeated continuously until the iteration is completed when the diameter l=60 nm, and finally determines the position of the fluorescent molecule, and the distance r between the fluorescent molecule and the nearest light spot center point in the iteration process can be expressed as:
Wherein, The representation is an estimate, the MLE representation is a maximum likelihood method, the maximum likelihood estimate/>The result of (a) is approximately expressed as polynomial distribution/>, of the probability of fluorescent molecules being closest to the spot center point in an iterationThe following formula is shown:
Wherein the method comprises the steps of Is a collection of photon numbers collected by the detector during the movement of the doughnut light spot; n is the total number of photons collected during a single iteration; n 0!…nk-1 -! Is/>The elements in the set represent the number of photons collected each time geometry I, II is moved in a single iteration; k is the number of times the central position of the geometric shapes I and II is moved by the control geometric shapes I and II of the spatial light modulators I and II in the single iteration process; p i is a vector parameter,/>Where lambda i denotes the wavelength of the background noise caused by geometry i and lambda bi denotes the wavelength of the background noise caused by geometry ii.
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