CN116593436A - Ultrafast super-resolution imaging method and system - Google Patents
Ultrafast super-resolution imaging method and system Download PDFInfo
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Abstract
The application discloses an ultrafast super-resolution imaging method, which comprises the following steps: focusing the laser on the sample surface after passing through the ferroelectric liquid crystal spatial light modulator, and collecting fluorescence emitted by the sample; generating a ferroelectric liquid crystal display diagram according to the exposure time and the view field, and splicing the display diagrams; according to the obtained ferroelectric liquid crystal splicing diagram, controlling the ferroelectric liquid crystal display and shutter opening to synchronously perform, and shooting to obtain an original diagram; and reconstructing according to the original image to obtain a super-resolution image. The application also discloses an ultrafast super-resolution imaging system. The optimized control time sequence can ensure that the structured light illumination super-resolution imaging can be performed in a large view field at high efficiency and high speed; the spatial filtering module provided by the application can perform spatial filtering at high speed, dynamically and accurately; the problems of low imaging speed, small field of view and inflexibility in the prior art are solved.
Description
Technical Field
The application belongs to the field of optical super-resolution microscopic imaging, and particularly relates to an ultrafast super-resolution imaging method and system.
Background
In the field of super-resolution fluorescence microscopy imaging, three technologies, namely a single molecule positioning technology, a stimulated radiation loss technology and a structured light illumination technology, are mainly available at present. The single-molecule positioning technology is a sub-nanometer level super-resolution imaging technology, and a plurality of sparsely distributed fluorescent images are subjected to data fusion, so that a high-resolution picture is obtained; but it can result in too slow an imaging speed. The stimulated radiation loss technology is to modulate the system point spread function through the excitation light and the loss light so as to realize super resolution. The structural light illumination technology is to shift the frequency of a high-frequency signal which cannot be acquired originally and has detail information into the system frequency domain through frequency shift, so that a high-resolution image is reproduced; compared with the two technologies, although the resolution of the structural light illumination technology is slightly lower, the structural light illumination technology does not need strong optical power, and the photo-bleaching phenomenon is avoided; and the super-resolution image reconstruction can be completed by only a few images, so that the imaging speed is greatly improved, and the method is suitable for research in the field of living cells.
The structural light illumination technology is characterized in that interference fringes in different directions and phases are generated on an imaging surface through translating and rotating a periodic physical grating, and high-frequency information is recovered through the frequency shift effect of moire fringes, so that a super-resolution picture is obtained; a structured light illumination super-resolution microscopic imaging system as provided in the publication CN106770147a, comprising: the super-resolution system comprises an illumination light source, a rotating structure light generator, a first converging lens, a spectroscope, an objective lens, an objective table, a sample, a second converging lens, digital imaging equipment and a computer, wherein an original image is processed through an imaging image reconstruction algorithm, and super-resolution can be realized by rotating the structure light stripe for 4 times without phase translation. Later, a structure light illumination based on ferroelectric liquid crystal is provided, and diffraction light of the ferroelectric liquid crystal can be regulated and controlled by regulating a display diagram of the ferroelectric liquid crystal, so that interference fringes generated on a sample surface can be regulated and controlled; therefore, only one ferroelectric liquid crystal is needed to realize the translation and rotation of stripes, and the imaging speed and the imaging precision are greatly improved; according to the super-resolution reconstruction method based on structured light illumination, structured light is generated by using a spatial light modulator, a sample to be detected of an object plane is modulated, original non-collectable high-frequency information is introduced after modulation, an image is firstly collected by using a CCD camera, structured light with phase shift pi is generated by using the spatial light modulator, a secondary collected image is obtained after modulation, a wide-field image is obtained by adding the primary collected image and the secondary collected image, the high-frequency information in the secondary collected image is separated by using the spectrum of the wide-field image and the spectrum generated by carrying out wiener filtering on the spectrum, filtering and spectrum movement operations are carried out, the high-frequency shift is returned to the original position, the spectrum is weighted and averaged by generalized wiener filtering, the spectrum with super-resolution is obtained, and finally the super-resolution image is obtained by carrying out inverse Fourier transform.
However, due to the problem of low diffraction efficiency of the ferroelectric liquid crystal, the structured light illumination based on the ferroelectric liquid crystal generally requires a long exposure time, the imaging speed is greatly affected by the exposure time, and the control timing generally used causes low illumination efficiency, further resulting in slow imaging speed.
In addition, in order to produce the desired interference fringes, the ferroelectric liquid crystal often requires spatial filtering of the spatial frequencies at which the interference occurs when the desired +1, -1 order coherent light is produced. However, the spatial filter generally used can only filter single frequency information, so that continuous spatial filtering cannot be realized, and the requirement of multicolor high-resolution imaging is difficult to meet.
Disclosure of Invention
Meanwhile, the application improves the defects of relatively more shutter opening time waste and low exposure efficiency during imaging, optimizes the control mode and improves the imaging speed and the illumination efficiency.
The application optimizes the structure of the space filter for the illumination of the multi-color structure light, and utilizes the space filter to carry out flexible space filtering on the Fourier plane information of the light with different wavelengths.
In order to achieve the above purpose, the application provides an ultrafast super-resolution imaging method and system, and the specific technical scheme is as follows:
an ultrafast super-resolution imaging method comprises the following steps:
focusing the laser on the sample surface after passing through the ferroelectric liquid crystal spatial light modulator, and collecting fluorescence emitted by the sample;
generating a ferroelectric liquid crystal display diagram according to the exposure time and the view field, and splicing the display diagrams;
according to the obtained ferroelectric liquid crystal splicing diagram, controlling the ferroelectric liquid crystal display and shutter opening to synchronously perform, and shooting to obtain an original diagram;
and reconstructing according to the original image to obtain a super-resolution image.
In the present application, the exposure time is determined in consideration of the fluorescence intensity of the sample. The exposure time is used to determine the system control timing, directly affecting the speed of imaging and the minimum field of view.
Determining that the required field of view and exposure time are similar, and determining the imaging speed of the system; but in addition to this, determining the field of view required for imaging also determines the maximum exposure time.
Preferably, the splicing of the ferroelectric liquid crystal display pictures is formed by cutting and splicing one or two original pictures.
Preferably, the picture cutting and splicing proportion is determined by the opening degree of a camera shutter when the ferroelectric liquid crystal displays pictures;
the black areas of the spliced display diagram represent the pictures where there is no display information; the spliced display diagram is from the original ferroelectric liquid crystal diagram, and all the clipping diagrams can be recombined into nine original ferroelectric liquid crystal display diagrams.
Preferably, the control of the ferroelectric liquid crystal display and the shutter opening are performed synchronously, and the exposure of the sample is controlled when the shutter of the camera is opened;
performing ferroelectric liquid crystal display once or twice in each shutter open period;
in the timing sequence, the ferroelectric liquid crystal display time should be ensured within the shutter open time of the camera.
Preferably, when shooting is carried out, the laser and the ferroelectric liquid crystal spatial light modulator are matched in parallel, so that the laser is ensured to be synchronously irradiated in the bitmap display period of the ferroelectric liquid crystal.
Preferably, the laser irradiation start time should be synchronized with the camera preset data transmission process.
Preferably, the bitmap display content of the ferroelectric liquid crystal is bitmap splicing of different directions and different phases; and dynamically adjusting the laser irradiation time length in the bitmap display period of the ferroelectric liquid crystal so as to adapt to different fluorescent sample shooting requirements.
In the present application, the preparation and switching period Ts of the ferroelectric liquid crystal is not necessarily equal to the bitmap display period Te of the ferroelectric liquid crystal; the ferroelectric liquid crystal bitmap display period Te can be reasonably configured according to the actual view field requirement, the exposure time is dynamically adjusted, and the average illumination intensity of each frame of data is increased;
further, the ferroelectric liquid crystal stitching bitmap T0 may be arbitrarily set according to the stripe shape, improving the imaging speed.
The time sequence control exposes the sample when the camera shutter is opened, so that the utilization rate of the exposure time of the system is improved, and the imaging quality is improved;
preferably, the time sequence control exposes the sample when the camera shutter is opened, so that the maximization of an imaging view field during high-speed imaging is ensured;
further, reconstructing the super-resolution picture with high real-time performance and long exposure time according to the obtained original picture.
The application also provides an ultrafast super-resolution imaging system, which comprises: the device comprises a light source module for emitting laser to illuminate a sample, a structural light modulation module for modulating the laser, a fluorescence detection module for projecting the laser to the sample and collecting fluorescence, and a time sequence control module for time sequence control and image processing; the structure light modulation module comprises a ferroelectric liquid crystal spatial light modulator, is loaded with a splice diagram displayed according to time sequence, and modulates an incident light beam;
the time sequence control module is used for:
generating a display diagram on the ferroelectric liquid crystal spatial light modulator according to the exposure time and the view field, and splicing the display diagram;
according to the obtained ferroelectric liquid crystal splicing diagram, controlling the ferroelectric liquid crystal display and shutter opening to synchronously perform, and shooting to obtain an original diagram;
and reconstructing according to the original image to obtain a super-resolution image.
In the application, the light source module comprises a laser light source and an acousto-optic modulator for controlling the laser intensity;
the structural light modulation module comprises a first half wave plate, a first polarization beam splitting cube, a second half wave plate, a ferroelectric liquid crystal spatial light modulator, an achromatic double-cemented lens, a first variable phase retarder, a second variable phase retarder, a quarter glass slide and a spatial filter which are sequentially arranged along a light path; the spatial filter consists of two mask plates and a motor, wherein the two mask plates have the same shape.
Preferably, the spatial filter filters +1, -1 order diffracted light of the ferroelectric liquid crystal;
the angle, direction and phase information of the +1, -1 diffraction light are controlled by a display diagram which is programmed in advance in the ferroelectric liquid crystal spatial light modulator;
the angle of the +1, -1 order diffracted light is changed according to different wavelength light to achieve maximization of each wavelength resolution;
the spatial filtering time is dynamically adjusted according to the filtering level, so that the matching of the position corresponding to the filtering hole of the spatial filter and +1 and-1 level spatial spectrum corresponding to the selected laser wavelength is ensured;
the spatial filter realizes filtering of different spatial frequencies through rotating the mask plate by the motor, can realize continuous filtering, and can realize filtering at any position of a two-dimensional plane;
the mask plate of the spatial filter is provided with six bending holes, so that the simultaneous filtering of six diffraction orders of ferroelectric liquid crystal can be realized.
The spatial filter can dynamically and continuously switch the position of spatial filtering at high speed;
before the sample is scanned, a monitoring camera is used for checking the stability and the fringe contrast of the system;
the monitoring camera is positioned on the tube mirror and the focal plane of the tube mirror, and monitors interference fringe information.
The application has the following beneficial effects:
1. the application realizes the high-speed and large-view-field imaging of the super-resolution imaging system based on the ferroelectric liquid crystal by adjusting the time coordination relation between the bitmap display of the ferroelectric liquid crystal and the camera shutter;
2. the application realizes the maximization of the sample exposure efficiency on the basis of rapid imaging by adjusting the time coordination relation between the ferroelectric liquid crystal bitmap display and the camera shutter;
3. the spatial filter can dynamically adjust the spatial filter height during spatial filtering, and has the characteristics of flexible adjustment and continuous adjustment relative to a mask plate with fixed holes, and is more suitable for spatial filtering based on ferroelectric liquid crystal;
4. according to the application, the spatial filter modules are placed back to back, so that the filter switching speed can be improved, and the high-speed stable characteristic of the filter is ensured;
5. according to the application, by optimally designing the spatial filtering module curve, high filtering precision can be ensured during dynamic filtering.
Drawings
FIG. 1 is a general method flow diagram of the present application;
FIG. 2 is a flow chart of a system control method of the present application;
FIG. 3 is a detailed view of the system control of the present application;
FIG. 4 is a schematic diagram of a spatial filter apparatus of the present application;
FIG. 5 is a schematic view of the overall apparatus and optical path of the present application;
FIG. 6 is a flow chart of the operation of the present application;
in the figure: 1. a laser light source; 2. an acousto-optic modulator; 3. a first half-wave plate; 4. a first polarization beam splitting cube; 5. a second half-wave plate; 6. a ferroelectric liquid crystal spatial light modulator; 7. achromatic double cemented lens; 8. a first variable phase retarder; 9. a second variable phase retarder; 10. a quarter slide; 11. a spatial filter; 12. a first tube mirror; 13. a second polarization beam splitting cube; 14. monitoring a camera; 15. a second tube mirror; 16. a reflecting mirror; 17. a dichroic mirror; 18. a microobjective; 19. a sample to be tested; 20. a light filter; 21. a third tube mirror; 22. an imaging camera; 23. a computer; 24. and a data acquisition card.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, however, the present application may be practiced in other ways than those described herein, and therefore the present application is not limited to the specific embodiments disclosed below.
Embodiments of the present application are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present application and should not be construed as limiting the application.
It should be noted that all directional indicators (such as up, down, left, right, front, and rear) in the embodiments of the present application are merely used to explain the relative positional relationship, movement conditions, etc. between the components in a specific posture (as shown in the drawings), and if the specific posture is changed, the directional indicators are changed accordingly.
Furthermore, descriptions such as those referred to herein as "first," "second," "a," and the like are provided for descriptive purposes only and are not to be construed as indicating or implying a relative importance or an implicit indication of the number of features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
In the present application, unless specifically stated and limited otherwise, the terms "connected," "affixed," and the like are to be construed broadly, and for example, "affixed" may be a fixed connection, a removable connection, or an integral body; can be mechanically or electrically connected; either directly or indirectly, through intermediaries, or both, may be in communication with each other or in interaction with each other, unless expressly defined otherwise. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art according to the specific circumstances.
Example 1:
as shown in fig. 1-6, an ultrafast super-resolution imaging method includes the steps of:
step 1: determining the exposure time and imaging field of view of the system, thereby determining the system T e ，T s And T r 。
Step 2: generating a bit map according to the system parameters, and splicing the bit maps;
in this embodiment, as shown in S21, the picture stitching is performed, and according to the calculated parameters, the basic binarized bitmaps (D11, D12, D13, D21, D22, D23, D31, D32, D33) are cut and stitched to form a stitched bitmap D0;
in this embodiment, the specific splicing process is as follows:
T e representing exposure time, T r Representing shutter open time, T s1 Representing the preparation time, T, of the ferroelectric liquid crystal display s2 Represents the switching time of ferroelectric liquid crystal display, wherein T s1 +T s2 ＝T s ；
At T e In this period, the field of view Hsize that the system can expose at the same time is determined by the height Hsize that the camera shutter has been opened, calculate the specific height of Hsize that can be obtained, and cut the ferroelectric liquid crystal bit map D11 to a height Hsize 2, obtain the first picture to be displayed of ferroelectric liquid crystal;
the second position to be displayed is positioned at the time when the second shutter is just opened, the visual fields which are far from the upper edge H2 and the lower edge H2 of the camera can be completely exposed, and a second picture to be displayed can be obtained after cutting;
the two clipping pictures can read out the complete D11 in one data reading period of the camera;
similarly, the third picture is positioned at the end time of the shutter opening, and in order to maximize the field of view, D12 is cut to a height H3, so that a third cut picture is obtained;
the fourth clipping diagram is special, and two bit bitmaps can be performed according to Hsize and H2: splicing D12 and D13;
the complete D12 map can be read out through the second read-out period;
the fifth clipping image is obtained by the method for obtaining the second clipping image;
in summary, all 14 splice graphs can be obtained.
Step 3: the time sequence of the system is controlled by the data acquisition card, so that the large view field, long exposure and high-speed imaging are ensured;
in this embodiment, the control timing of the camera is specifically as follows in step S21:
according to the preset view field, opening the camera shutter, and performing one or two ferroelectric liquid crystal bitmap display in each camera shutter opening time.
The irradiation amplitude of the laser is adjusted according to the required exposure time in the ferroelectric liquid crystal bitmap display period, and the synchronization of the laser and the ferroelectric liquid crystal display period is maintained.
The data acquisition card is used as a main control, and the laser controlled by the acousto-optic modulator and the ferroelectric liquid crystal spatial light modulator are matched in parallel, so that the laser is ensured to be synchronously irradiated in the bitmap display period of the ferroelectric liquid crystal.
In the bitmap display period of the ferroelectric liquid crystal, the laser irradiation time length can be dynamically adjusted to adapt to different fluorescent sample shooting requirements; the bitmap display content of the ferroelectric liquid crystal is bitmap splicing of different directions and different phases.
In this embodiment, the laser irradiation period is guaranteed to be consistent with the stable switching state of the polarization control device; the laser irradiation period should be synchronized with the camera preset data transmission time. The camera trigger mode should be a synchronous trigger mode and not always at global exposure time; the bitmap display period of the ferroelectric liquid crystal is always within the shutter open period of the camera.
Preparation and switching period T of ferroelectric liquid crystal s Not necessarily equal to the bit-mapped display period T of ferroelectric liquid crystal e Ferroelectric liquid crystal bitmap display period T e The exposure time can be dynamically adjusted by reasonable configuration of the actual field of view, and the average illumination intensity of each frame of data is increased.
In this embodiment, the ferroelectric liquid crystal stitching bitmap (T0) may be arbitrarily set according to the stripe shape, so as to improve the imaging speed. The illumination efficiency η for a commonly used control timing can be expressed as:
wherein T is e Represents exposure time, T r Representing the shutter open time of the camera and also representing the data read time, T s Representing the sum of the preparation time required for the ferroelectric liquid crystal spatial light modulator to display a bitmap and the switching time required for the display to end; when large field imaging is required, the exposure efficiency η is very small and the light capacity is very underutilized;
in contrast, the system controls the time sequence in the continuous imaging of a large field of view, namely T s ＜＜T r Reasonable design T r Such that:
wherein when T r ＞T e To ensure uniformity of exposure, the laser irradiation time is less than T e The method comprises the steps of carrying out a first treatment on the surface of the When T is r ＜T e To ensure uniformity of exposure, the laser irradiation time is longer than T e ；
The efficiency η' can be greatly improved:
η′；50％＞η
the time for opening the camera shutter is reasonably utilized, the time utilization rate is ensured, and the exposure efficiency eta is improved;
the sampling time T' of nine graphs is obviously shortened compared with the sampling time T used by a common time sequence while realizing the higher efficiency eta;
T＝9×(T r +T e )
T′＝9×(T r )＜T
d11, D12, D13 are respectively three different phase diagrams of direction, and by combining the picture concatenation of D0, the sequential display of three different phase diagrams of three directions is easy to realize: d11, D12, D13, D21, D22, D23, D31, D32, D33; so as to increase the imaging speed by three times again on the basis of the time sequence by combining algorithms such as staggered reconstruction and the like. Likewise, a seven-image, five-image reconstructed spatial light modulator bitmap stitching is easily implemented for higher imaging.
Step 4: reconstructing the image to obtain a super-resolution image. The original image acquired by the camera can be reconstructed by an algorithm to obtain a large-view-field high-speed super-resolution image.
In this embodiment, the specific operation flows of steps 1 to 4 are shown in fig. 6.
The application also provides a spatial filter, which can realize continuous, high-speed and high-precision filtering in the frequency domain based on the hardware system and the time sequence;
when using super-resolution microscopy imaging systems based on structured light illumination, spatial filtering of the spectral planes is required. For ferroelectric liquid crystals, spatial filtering of +1, -1 order light is required; the angle, direction and phase information of +1, -1 order diffraction light are controlled by bit map which is programmed in advance in the ferroelectric liquid crystal spatial light modulator; the angle of the +1, -1 order diffracted light varies according to the different wavelengths of light, and flexible spatial filtering is performed in combination with the spatial filter 11 according to the present application to maximize the resolution of each wavelength.
In this embodiment, the spatial filter structure is as follows:
the spatial filter 11 is composed of two mask plates 25 of fixed shape and a motor;
the two mask plates 25 with fixed shapes keep a coaxial state back to back, so that the spatial filtering function is exerted together, the filtering precision is improved, and the switching speed of different filtering processes can be ensured;
compared with a mask plate with fixed hole digging, the structure can ensure the continuous switching of different filtering frequencies of a frequency spectrum surface;
the slope of a punching curve in the space mask 25 is optimally designed, so that the deformation of a formed filtering hole is minimum in the changing process;
when the two masks 25 are rotated relatively, the filter aperture will shift, as shown by spatial filter module 26 and spatial filter module 27; when the two masks are relatively static and jointly move along a certain direction, the spatial filtering of any position in the whole plane can be realized;
the structure can realize random frequency space filtering in six directions at most, and is perfectly suitable for the requirements of a structured light illumination microscopic system; if a through hole is drilled at the 0-frequency light, seven paths of light can be filtered simultaneously.
The application also proposes a device for monitoring the contrast of interference fringes by using a monitoring camera, wherein the monitoring camera is positioned on the focal planes of the tube mirror 12 and the tube mirror 15 and is used for monitoring the interference fringe information; the imaging stability is ensured.
Example 2:
as shown in fig. 1-6, an ultrafast super-resolution imaging system, comprising: the device comprises a light source module, a structural light modulation and monitoring module, a fluorescence detection module and a time sequence control module.
The laser sequentially passes through the light source module, the structural light modulation and monitoring module and the fluorescence detection module to reach the camera image acquisition surface to be acquired.
The light source module comprises a laser light source 1 and an acousto-optic modulator 2; the acousto-optic modulator 2 is used for controlling the laser intensity;
the structural light modulation and monitoring module comprises a first half wave plate 3, a first polarization beam splitting cube 4, a second half wave plate 5, a ferroelectric liquid crystal spatial light modulator 6, an achromatic double-cemented lens 7, a first variable phase retarder 8, a second variable phase retarder 9, a quarter glass 10, a spatial filter 11, a first tube mirror 12, a second polarization beam splitting cube 13, a monitoring camera 14, a second tube mirror 15 and a reflecting mirror 16; the first half wave plate 3 is used for regulating and controlling the light intensity entering the first polarization beam splitting cube 4; the first polarization beam splitting cube 4, the second half wave plate 5 and the ferroelectric liquid crystal spatial light modulator 6 are combined to generate diffracted light with different directions and different phases; the first variable phase retarder 8, the second variable phase retarder 9 and the quarter-slide 10 are used for regulating and controlling the polarization directions of diffracted lights in different directions; the spatial filter 11 is used for spatial filtering, and only allows light diffracted by the ferroelectric liquid crystal spatial light modulator 6 at a specific angle to pass through, and blocks other diffraction orders; the achromatic double-cemented lens 7 and the first tube mirror 12 form a 4f system; the monitoring camera 14 is used for monitoring interference fringe information of the intermediate image plane.
The fluorescence detection module comprises a dichroic mirror 17, a microscope objective 18, a sample 19 to be detected, an optical filter 20, a third tube mirror 21 and an imaging camera 22; the dichroic mirror 17 is used to separate the laser and fluorescence signals; the microscope objective 18 and the third tube mirror 21 cooperate to image the fluorescent signal at the imaging camera 22.
The timing control module includes: computer 23, data acquisition card 24, the electrical connection between computer 23 and the data acquisition card 24.
In this embodiment, the matching manner of the hardware and the imaging method is specifically as follows:
preparing an observation sample, and controlling the laser light source 1, the acousto-optic modulator 2, the ferroelectric liquid crystal spatial light modulator 6, the first variable phase retarder 8, the second variable phase retarder 9, the spatial filter 11 and the imaging camera 22 to be turned on and preheated by using the computer 23, and turning on the monitoring camera 14 if streaking and system monitoring are performed;
determining exposure time, determining field of view, and controlling the imaging camera 22 to set corresponding exposure time by the computer 23; controlling the ferroelectric liquid crystal spatial light modulator 6 by the computer 23 to set an appropriate imaging timing; generating a corresponding ferroelectric liquid crystal spatial light modulator 6 original image; splicing the display diagrams according to the exposure time and the time sequence of the ferroelectric liquid crystal spatial light modulator 6;
picture splicing is shown in fig. 2, and according to the calculated parameters, a spliced bit bitmap D0 is formed by cutting and splicing basic binary bit bitmaps (D11, D12, D13, D21, D22, D23, D31, D32 and D33);
in this embodiment, the specific splicing process is as follows:
T e representing exposure time, T r Representing shutter open time, T s1 Representing the preparation time, T, of the ferroelectric liquid crystal display s2 Represents the switching time of ferroelectric liquid crystal display, wherein T s1 +T s2 ＝T s ；
At T e During this time, the field of view Hsize that the system is able to simultaneously expose is already open by the camera shutterDetermining the height Hsize, calculating the specific height of Hsize, and cutting the ferroelectric liquid crystal bit map D11 with the height of Hsize being 2 to obtain a first picture to be displayed of ferroelectric liquid crystal;
the second position to be displayed is positioned at the time when the second shutter is just opened, the visual fields which are far from the upper edge H2 and the lower edge H2 of the camera can be completely exposed, and a second picture to be displayed can be obtained after cutting;
the two clipping pictures can read out the complete D11 in one data reading period of the camera;
similarly, the third picture is positioned at the end time of the shutter opening, and in order to maximize the field of view, D12 is cut to a height H3, so that a third cut picture is obtained;
the fourth clipping diagram is special, and two bit bitmaps can be performed according to Hsize and H2: splicing D12 and D13;
the complete D12 map can be read out through the second read-out period;
the fifth clipping image is obtained by the method for obtaining the second clipping image;
in summary, all 14 tiles (D0) can be obtained.
Finally, preloading 14 spliced pictures on an internal storage of the ferroelectric liquid crystal spatial light modulator 6;
the acousto-optic modulator 2 is gated and the laser light is emitted.
The laser is transmitted through the first polarization beam splitting cube 4 after the polarization direction of the laser is adjusted by the first half-wave plate 3, is further adjusted by the second half-wave plate 5, irradiates onto the ferroelectric liquid crystal spatial light modulator 6, and reflects specific diffracted light after modulation of the spatial light modulator.
As shown in fig. 2, the ferroelectric liquid crystal spatial light modulator 6 first displays the first mosaic D0, and switches the mosaic at the end of each display period.
The diffracted light passes through the first polarization beam splitting cube 4 after the polarization direction of the diffracted light is regulated by the second half wave plate 5, passes through the achromatic double-cemented lens 7, and then is modulated in the polarization direction by the first variable phase retarder 8, the second variable phase retarder 9 and the quarter-wave plate 10, so that the polarization direction of +1st-1st-order light is controlled to be consistent, and the interference contrast of the two beams of light is maximized.
The diffracted light reaches the spatial filter 11 positioned at the back focal plane position after polarization modulation, and the light of other diffraction orders is filtered by the spatial filter 11, so that only +1 and-1 orders of light pass through.
The spatial filter 11 is adjusted as follows: when the two masks 25 are rotated relatively, the filter aperture will shift, as shown by spatial filter module 26 and spatial filter module 27; when the two masks are relatively static and move along a certain direction together, the spatial filtering at any position in the whole plane can be realized.
The filtered diffracted light reaches the first tube mirror 12 and then enters the second polarization beam splitting cube 13, and part of the diffracted light is reflected to the monitoring camera 14; the other part of diffracted light is transmitted through the second polarization beam splitting cube 13 and the second tube mirror 15 in sequence, reflected by the reflecting mirror 16 and the dichroic mirror 17, and transmitted through the microscope objective 18 to be focused on the sample surface.
If streaking and system monitoring is performed, then monitoring and judgment can be performed by the monitoring camera 14 at this time.
Fluorescence excited by the sample surface reversely passes through the microscope objective 18, the dichroic mirror 17, the optical filter 20 and the third tube mirror 21 and then reaches the imaging camera 22;
the bitmap display period of the ferroelectric liquid crystal spatial light modulator 6 is always within the shutter open period of the imaging camera 22, and the specific relationship between the two periods satisfies:
wherein T is e Represents exposure time, T r Representing the shutter open time of the camera and also representing the data read time, T s Representing the sum of the preparation time required for the ferroelectric liquid crystal spatial light modulator to display a bitmap and the switching time required for the display to end.
In combination with the control sequence shown in fig. 2, the data acquisition card 24 controls the ferroelectric liquid crystal spatial light modulator 6 to display and switch the splicing diagram, and the splicing diagrams of D0-D14 are sequentially displayed; controlling the small hole direction and small Kong Gaodi of the spatial filter 11 to be matched with the level of the spatial filtering; and keep the first variable phase retarder 8, the second variable phase retarder 9 of the synchronous control change in each display period, in order to achieve the purpose of polarization control; simultaneously keeping the shutter of the imaging camera 22 open and synchronous in each display period, and shooting a sample to obtain an original image shot by the sample;
and (3) cycling the previous step to obtain all original pictures, and reconstructing the original pictures to obtain super-resolution pictures.
The ultra-fast super-resolution imaging system provided by the embodiment solves the problems of low imaging speed, small field of view and inflexibility in the prior art. The spatial filtering module provided by the application can perform spatial filtering at high speed, dynamically and accurately; the optimized control time sequence can ensure that the structured light illumination super-resolution imaging can be performed in a large view field at high efficiency and high speed.
The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the application are intended to be included within the scope of the application.
Claims (10)
1. An ultrafast super-resolution imaging method, comprising:
focusing the laser on the sample surface after passing through the ferroelectric liquid crystal spatial light modulator, and collecting fluorescence emitted by the sample;
generating a ferroelectric liquid crystal display diagram according to the exposure time and the view field, and splicing the display diagrams;
according to the obtained ferroelectric liquid crystal splicing diagram, controlling the ferroelectric liquid crystal display and shutter opening to synchronously perform, and shooting to obtain an original diagram;
and reconstructing according to the original image to obtain a super-resolution image.
2. The ultra-fast super-resolution imaging method according to claim 1, wherein the stitching of the ferroelectric liquid crystal display pictures is formed by stitching one or two original pictures.
3. The ultra-fast super-resolution imaging method according to claim 2, wherein the picture cropping and stitching ratio is determined by an opening degree of a camera shutter when the picture is displayed by the ferroelectric liquid crystal;
the black areas of the spliced display diagram represent the pictures where there is no display information; the spliced display diagram is from the original ferroelectric liquid crystal diagram, and all the clipping diagrams can be recombined into nine original ferroelectric liquid crystal display diagrams.
4. The ultra-fast super-resolution imaging method according to claim 1, wherein the controlling of the ferroelectric liquid crystal display and the shutter opening are performed simultaneously, and the exposing of the sample is controlled when the shutter of the camera is opened;
performing ferroelectric liquid crystal display once or twice in each shutter open period;
in the timing sequence, the ferroelectric liquid crystal display time should be ensured within the shutter open time of the camera.
5. The ultra-fast super-resolution imaging method according to claim 4, wherein the laser and the ferroelectric liquid crystal spatial light modulator are matched in parallel during shooting, so that the laser is ensured to be synchronously irradiated in a bitmap display period of the ferroelectric liquid crystal.
6. The ultra-fast super-resolution imaging method according to claim 4, wherein the laser irradiation start time is synchronized with a camera preset data transmission process.
7. The ultra-fast super-resolution imaging method according to claim 4, wherein the bitmap display contents of the ferroelectric liquid crystal are bitmap stitching of different directions and phases;
and dynamically adjusting the laser irradiation time length in the bitmap display period of the ferroelectric liquid crystal so as to adapt to different fluorescent sample shooting requirements.
8. An ultrafast super-resolution imaging system, comprising: the device comprises a light source module for emitting laser to illuminate a sample, a structural light modulation module for modulating the laser, a fluorescence detection module for projecting the laser to the sample and collecting fluorescence, and a time sequence control module for time sequence control and image processing; the method is characterized in that: the structure light modulation module comprises a ferroelectric liquid crystal spatial light modulator (6) loaded with a splice graph displayed according to time sequence, and modulates an incident light beam;
the time sequence control module is used for:
generating a display diagram on the ferroelectric liquid crystal spatial light modulator (6) according to the exposure time and the view field, and splicing the display diagram;
according to the obtained ferroelectric liquid crystal splicing diagram, controlling the ferroelectric liquid crystal display and shutter opening to synchronously perform, and shooting to obtain an original diagram;
and reconstructing according to the original image to obtain a super-resolution image.
9. The ultra-fast super-resolution imaging system according to claim 8, wherein the light source module comprises a laser light source (1) and an acousto-optic modulator (2) for controlling the laser intensity;
the structural light modulation module comprises a first half wave plate (3), a first polarization beam splitting cube (4), a second half wave plate (5), a ferroelectric liquid crystal spatial light modulator (6), an achromatic double-cemented lens (7), a first variable phase retarder (8), a second variable phase retarder (9), a quarter glass slide (10) and a spatial filter (11) which are sequentially arranged along a light path; the spatial filter (11) consists of two mask plates and a motor, wherein the two mask plates have the same shape.
10. The ultra-fast super-resolution imaging system according to claim 9, wherein the spatial filter (11) filters +1, -1 order diffracted light of ferroelectric liquid crystals;
the angle, direction and phase information of the +1, -1 diffraction light are controlled by a preprogrammed display diagram in the ferroelectric liquid crystal spatial light modulator;
the angle of the +1, -1 order diffracted light is changed according to different wavelength light to achieve maximization of each wavelength resolution;
the spatial filtering time is dynamically adjusted according to the filtering level, so that the matching of the position corresponding to the filtering hole of the spatial filter and +1 and-1 level spatial spectrum corresponding to the selected laser wavelength is ensured;
the spatial filter realizes filtering of different spatial frequencies by rotating the mask plate through a motor;
the mask plate of the spatial filter is provided with six bending holes, so that the simultaneous filtering of six diffraction orders of ferroelectric liquid crystal can be realized.
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