Structured light illumination system, structured light illumination method and super-resolution optical system

Abstract

A structured light illumination system, a structured light illumination method and a super-resolution optical system, the structured light illumination system comprising: at least two light sources (1), the light sources (1) being used for generating light beams of a preset wavelength; the at least two optical gratings (2) are respectively positioned on the light paths of the at least two light sources (1), and the optical gratings (2) are used for generating interference fringes; the grating (2) is arranged on the displacement table (3), and the displacement table (3) is used for moving the grating (2) to perform phase switching; and a light beam converging and guiding unit (4) at which the light paths converge, the light beam converging and guiding unit (4) being used for guiding the light beams to converge to the object space. According to the structured light illumination system, rapid angle and phase switching can be carried out on structured light stripes, and the imaging speed is improved, so that the flux of an optical system is improved, and the imaging cost is greatly reduced while the resolution of the optical system is improved.

Description

Structured light illumination system, structured light illumination method, and super-resolution optical system Technical Field

[0001] The present invention relates to the field of optical imaging technology, and in particular to a structured light illumination system, a structured light illumination method and a super-resolution optical system. Background Art

[0002] Gene sequencing technology is widely used in various research areas of life sciences and medicine, including various genomics, the etiology of complex diseases, prenatal diagnosis, and personalized drug treatment. A common gene sequencing method uses a high-resolution fluorescence microscopy system, where fluorescent groups, upon being excited by lasers of different wavelengths, emit fluorescence of varying wavelengths. Sequencing is achieved by photographing the sequencing chip.

[0003] The resolution of current super-resolution optical imaging systems can be reduced to below 100 nanometers. Among the common super-resolution imaging methods currently on the market, the stimulated emission depletion method requires high excitation light intensity and is not suitable for long sequencing. At the same time, its point scanning characteristics make it more suitable for rapid imaging of small-area samples. Photoactivated localization microscopy and stochastic optical reconstruction microscopy can achieve very high resolution, but due to their single-molecule localization characteristics, the acquisition time of each super-resolution image is basically on the order of minutes, and the sequencing speed is slow. Structured illumination microscopy and spinning disk confocal microscopy based on pixel redistribution can meet the requirements of large-scale, high-speed, and high-resolution imaging, and have no specific requirements for fluorescent dyes. The required light intensity is also relatively low, and they have been combined with existing sequencing technologies.

[0004] There are currently two methods for generating structured light. One is projection, which uses a spatial light modulator (SLM) or a digital microlens array (DMD) to project the interference fringe pattern onto the object surface. The advantage of this method is its fast modulation speed, but the resolution of SLM and DMD is generally below 1920*1200, corresponding to an object field of view of only tens to hundreds of microns. At the same time, the damage threshold of SLM is low and it cannot withstand high-power laser irradiation. The other is grating interferometry, which uses a grating to separate two beams of light, which are then combined on the object side through a certain optical design to form interference fringes. Grating interferometry is not limited by size, and its corresponding object field of view can reach above the millimeter level. At the same time, according to different resolution requirements, interference fringes with a period of hundreds of nanometers can be obtained. Based on these characteristics, grating interferometry is particularly suitable for high-throughput needs such as gene sequencing.

[0005] Currently, the cost of gene sequencing is mainly concentrated on sequencing reagents. By improving the resolution of the imaging system, the amount of sequencing data per unit area can be increased, and the utilization rate of sequencing reagents can be improved. For the same amount of data, the reagent cost will be greatly reduced by improving the resolution of the system. Since the cost of reagent consumables is inversely proportional to the square of the sample density, how to use super-resolution imaging technology to increase the sample density of sequencing chips and further reduce the cost of gene sequencing is an urgent problem to be solved. The field of view of existing super-resolution imaging technology is mostly tens to hundreds of microns, and a rotating stage is required to rotate the grating to switch different structured light directions. However, the speed of the rotating stage is slow, and it generally takes hundreds of milliseconds to rotate 90°, resulting in slow imaging speed and low information throughput, which makes it difficult to meet the increasingly high high-throughput sequencing requirements on the market.

[0006] Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects of the prior art such as small imaging field of view, slow speed and low information flux, and to provide a structured light illumination system, a structured light illumination method and a super-resolution optical system.

[0008] The present invention solves the above technical problems through the following technical solutions:

[0009] The present invention provides a structured light illumination system, comprising:

[0010] at least two light sources, each configured to generate a light beam of a predetermined wavelength;

[0011] At least two gratings, respectively located on the optical paths of the at least two light sources, the gratings being used to generate interference fringes;

[0012] a translation stage, the grating being arranged on the translation stage, and the translation stage being used to move the grating to perform phase switching;

[0013] A light beam merging and guiding unit, wherein the light paths converge at the light beam merging and guiding unit, and the light beam merging and guiding unit is used to guide the light beams to merge into the object space.

[0014] Preferably, the light beam merging and guiding unit comprises:

[0015] A galvanometer, wherein the optical paths intersect at the galvanometer, and the galvanometer is used to reflect the light beams to the object space by swinging at at least two preset angles.

[0016] Preferably, the structured light illumination system comprises:

[0017] a first light source and a second light source, wherein the first light source and the second light source are used to generate light beams with different polarization directions;

[0018] The light beam merging and guiding unit comprises:

[0019] A polarization beam combiner, wherein the optical paths intersect at the polarization beam combiner, and the polarization beam combiner is used to reflect the light beam of the first light source to the object space, and the light beam of the second light source passes through the polarization beam combiner to reach the object space.

[0020] Preferably, the at least two gratings are arranged in directions orthogonal to each other to generate orthogonal interference fringes; and / or,

[0021] The at least two gratings include a modulated grating having a high transmittance at a preselected energy level.

[0022] Preferably, the light source is specifically configured to generate light beams of at least two different preset wavelengths; and / or,

[0023] The structured light illumination system further comprises:

[0024] At least two collimating lenses are respectively located on the optical path, and the light beam reaches the grating after being collimated by the collimating lenses.

[0025] Preferably, the structured light illumination system further comprises:

[0026] At least two filtering modules are respectively located on the optical path and are used to filter out light beams outside of a preselected energy level so that the light beams of the preselected energy level can reach the light beam merging and guiding unit.

[0027] Preferably, the filtering module includes:

[0028] The first lens and the second lens are used to form a first 4f system; the grating is located on the front focal plane of the first 4f system, and the beam merging and guiding unit is located on the back focal plane of the first 4f system;

[0029] The energy level filter is located on the spectrum plane of the first 4f system and is used to filter out light beams outside the preselected energy level so that the light beams of the preselected energy level can reach the light beam merging and guiding unit.

[0030] Preferably, the structured light illumination system further comprises:

[0031] The third lens and the fourth lens are used to form a second 4f system; the light beam merging and guiding unit is located on the front focal plane of the second 4f system, and the object plane is located on the back focal plane of the second 4f system.

[0032] Preferably, the structured light illumination system further comprises:

[0033] a dichroic mirror, located on the optical path between the third lens and the fourth lens, for reflecting the structured light emitted by the third lens toward the object;

[0034] The object-side signal generated by the structured light illumination is received after passing through the dichroic mirror.

[0035] The present invention further provides a structured light illumination method, which is applied to the structured light illumination system described above, and includes:

[0036] Controlling the translation stage to move a first grating of the at least two gratings to a first preset position, and causing the first light source of the at least two light sources to emit light beams, which are guided by a beam merging and guiding unit to merge into an object space to generate structured light of a first direction and a first phase;

[0037] Controlling the translation stage to sequentially move the first grating to other preset positions, the first light source emits light beams, which are guided by the light beam merging and guiding unit to merge into the object space to generate structured light of other phases in the first direction;

[0038] The translation stage is controlled to sequentially move the other gratings of the at least two gratings to preset positions, and the other light sources of the at least two light sources emit light beams, which are guided by the light beam merging and guiding unit to merge into the object space to generate structured light in other directions.

[0039] The present invention further provides a super-resolution optical system, comprising a substrate, an imaging module, an auto-focus module, and an illumination module; the imaging module, the auto-focus module, and the illumination module are fixed on the substrate; the illumination module adopts the structured light illumination system described above;

[0040] The auto-focus module is coupled to the lighting module via a galvanometer, and the auto-focus module is used to drive the objective lens to achieve auto-focus;

[0041] The lighting module is used to emit a lighting structure light beam which is projected onto the surface of the sample to be photographed through the galvanometer and the objective lens;

[0042] The imaging module is used to receive the optical signal generated by the sample to be photographed after being irradiated by the illumination structure light beam; the optical signal is divided into several optical signals with different wavelengths after passing through the objective lens and several dichroic mirrors.

[0043] The present invention also provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the structured light illumination method as described above when executing the computer program.

[0044] The present invention also provides a computer-readable medium having computer instructions stored thereon, which implement the structured light illumination method described above when executed by a processor.

[0045] The positive progress effect of the present invention is:

[0046] The structured light illumination system provided by the present invention utilizes a translation stage to move a grating to rapidly switch the phase of structured light stripes. A beam merging and guiding unit guides light beams of different angles to converge in the object space to rapidly switch the angles of the structured light stripes, thereby increasing the imaging speed and thus the throughput of the optical system, improving the resolution of the optical system while significantly reducing the imaging cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] To more clearly illustrate the technical solutions of the embodiments of this specification, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are merely examples or embodiments of this specification. For those skilled in the art, it is possible to apply this specification to other similar scenarios based on these drawings without inventive effort.

[0048] FIG1 is a first structural diagram of a structured light illumination system in embodiment 1 of the present invention.

[0049] Figure 2 shows the original OTF spectrum of the structured light super-resolution imaging principle.

[0050] Figure 3 shows the extended OTF spectrum of the structured light super-resolution imaging principle.

[0051] FIG4 is a second structural diagram of the structured light illumination system in Embodiment 2 of the present invention.

[0052] FIG5 is a front view of the structured light illumination system in Example 2 of the present invention.

[0053] FIG6 is a perspective view of the structured light illumination system in Example 2 of the present invention.

[0054] FIG7 is a schematic structural diagram of the energy level filter in Example 2 of the present invention.

[0055] FIG8 is a third structural diagram of the structured light illumination system in Embodiment 2 of the present invention.

[0056] FIG9 is a flow chart of the structured light illumination method in embodiment 3 of the present invention.

[0057] FIG10 is a timing flow chart of a structured light illumination system using a high-speed galvanometer in Example 3 of the present invention.

[0058] FIG11 is a front view of the structure of the super-resolution optical system in Example 4 of the present invention.

[0059] FIG12 is a perspective view of the first structure of the super-resolution optical system in Example 4 of the present invention.

[0060] FIG13 is a perspective view of a second structure of the super-resolution optical system in Example 4 of the present invention.

[0061] FIG14 is a schematic diagram showing the optical path structure principle of the super-resolution optical system in Example 4 of the present invention.

[0062] FIG15 is a first structural diagram of an imaging module of a super-resolution optical system in Example 4 of the present invention.

[0063] FIG16 is a second structural diagram of the imaging module of the super-resolution optical system in Example 4 of the present invention.

[0064] FIG17 is a schematic structural diagram of an electronic device in Example 5 of the present invention. DETAILED DESCRIPTION

[0065] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0066] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various places herein does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0067] As used herein, unless the context clearly indicates otherwise, the terms "a," "an," "an," and / or "the" are not intended to refer to the singular but may include the plural. Generally speaking, the terms "include" and "comprise" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include additional steps or elements.

[0068] The definitions of "first" and "second" herein, and the descriptions "first," "second," etc., are provided for illustrative purposes only and are not intended to be sequential or to limit the number of devices herein. They should not be construed as limiting this disclosure. For example, a first element could be referred to as a second element without departing from the scope of this disclosure. Similarly, a second element could be referred to as a first element.

[0069] Flowcharts are used herein to illustrate the operations performed by the systems according to the embodiments of the present invention. It should be understood that the preceding or following operations do not necessarily need to be performed in exact order. Instead, the steps may be processed in reverse order or simultaneously. Furthermore, other operations may be added to these processes, or one or more operations may be removed from these processes.

[0070] Example 1

[0071] Please refer to Figure 1, which is a first structural diagram of the structured light illumination system in this embodiment. Specifically, as shown in Figure 1, the structured light illumination system includes:

[0072] At least two light sources 1, the light sources are used to generate light beams of preset wavelengths;

[0073] At least two gratings 2, respectively located on the optical paths of at least two light sources 1, the gratings 2 being used to generate interference fringes;

[0074] a translation stage 3 on which the grating 2 is disposed, and for moving the grating 2 to perform phase switching;

[0075] The light beam merging and guiding unit 4 , where the light paths converge, is used to guide the light beams to merge into the object space.

[0076] The structured light illumination system provided in this embodiment utilizes a translation stage to move a grating to rapidly switch the phase of the structured light stripes. A beam converging and guiding unit guides light beams of different angles to converge in the object space to rapidly switch the angles of the structured light stripes. This improves the imaging speed, thereby increasing the throughput of the optical system, improving the resolution of the optical system, and significantly reducing the imaging cost.

[0077] Example 2

[0078] The structured light illumination system of this embodiment is a further improvement of the embodiment 1. Specifically:

[0079] In an optional embodiment, the light beam merging and guiding unit includes:

[0080] The optical path intersects the galvanometer, which is used to reflect the light beam to the object space by swinging at at least two preset angles. Specifically, the high-speed galvanometer can achieve rapid switching of the structured light direction by swinging at a small angle at high speed.

[0081] In another optional embodiment, the structured light illumination system includes:

[0082] a first light source and a second light source, the first light source and the second light source are used to generate light beams with different polarization directions;

[0083] The beam combining and guiding unit includes:

[0084] Polarization beam combiner: the optical paths intersect at the polarization beam combiner, which is used to reflect the light beam from the first light source to the object space, and the light beam from the second light source passes through the polarization beam combiner to reach the object space.

[0085] Specifically, two illumination structured lights in different directions use light sources with different polarization states, and then are combined using a polarization beam combiner (PBC) to improve the system light efficiency.

[0086] In this embodiment, the light source is specifically configured to generate at least two light beams of different preset wavelengths; the at least two gratings are arranged in directions orthogonal to each other to generate orthogonal interference fringes; and the at least two gratings include modulated gratings, which have high transmittance at a preselected energy level.

[0087] The structured light illumination system also includes:

[0088] At least two collimating lenses are respectively located on the optical path, and the light beam reaches the grating after being collimated by the collimating lenses.

[0089] In an optional embodiment, the structured light illumination system further includes:

[0090] At least two filtering modules are respectively located on the optical path and are used to filter out light beams outside the preselected energy level so that the light beams of the preselected energy level can reach the light beam merging and guiding unit.

[0091] Specifically, the filtering module includes:

[0092] The first lens and the second lens are used to form a first 4f system; the grating is located on the front focal plane of the first 4f system, and the galvanometer is located on the back focal plane of the first 4f system;

[0093] The energy level filter is located on the spectrum plane of the first 4f system and is used to filter out the light beam outside the preselected energy level so that the light beam of the preselected energy level reaches the galvanometer.

[0094] In this embodiment, the structured light illumination system further includes:

[0095] The third lens and the fourth lens are used to form a second 4f system; the light beam merging and guiding unit is located on the front focal plane of the second 4f system, and the object plane is located on the back focal plane of the second 4f system.

[0096] In addition, the structured light illumination system also includes:

[0097] The dichroic mirror is located on the optical path between the third lens and the fourth lens, and is used to reflect the structured light emitted by the third lens toward the object. The object signal generated by the structured light is received after passing through the dichroic mirror.

[0098] For optical microscopy systems, the point spread function (PSF) can be characterized by a Bessel function, which is represented in the frequency domain by the optical transfer function (OTF). As shown in Figure 2, the OTF in the frequency domain is equivalent to a spectral filter. The size of the OTF determines the resolution of the optical system. Low-frequency information within the circle can be collected by the optical system, while high-frequency information outside the circle is filtered out. As shown in Figure 3, when using structured light illumination with a frequency of k, the image information collected by the system will contain information at three different frequencies: k+k0, k, and k-k0. The super-resolution reconstruction algorithm then calculates the information at these three frequencies to obtain an expanded frequency domain space, thereby improving the system's resolution.

[0099] Based on the aforementioned structured light super-resolution imaging principle, achieving super-resolution imaging requires acquiring six structured light images from two directions and three phases. The following example further illustrates the structured light illumination system by capturing six structured light images from two directions and three phases.

[0100] Please refer to Figure 4, which is a second schematic diagram of the structured light illumination system in this embodiment. Figure 4 shows a V-shaped structured light illumination system, in which two different directions of structured light are generated by the two sides of the V-shaped illumination system. At the same time, a high-speed galvanometer is installed at the vertex of the V-shaped illumination system. The high-speed and small-angle swing of the galvanometer can achieve rapid switching of the structured light direction. The specific technical solution is described as follows:

[0101] As shown in FIG4 , elements 101 to 106 constitute a first illumination optical path, and elements 111 to 116 constitute a second illumination optical path. The two illumination optical paths have similar structures, except that the gratings are installed in different directions, forming a 90-degree angle with each other. Since the two optical paths have similar structures, descriptions such as “light source 101 / 111” used below indicate that the two light sources have the same characteristics; descriptions such as “first light source 101, second light source 111” used below indicate that the two light sources have different functions or properties.

[0102] Light source 101 / 111 is a laser with an optical fiber output as an illumination excitation light source. Light source 101 / 111 generally includes two laser wavelengths, 532nm (nanometers) and 660nm. The two wavelengths share one optical fiber output, and the power of each wavelength is 4W (watts).

[0103] The parallel light emitted by the light source 101 / 111 is collimated by the lens 102 / 112 and reaches the grating 103 / 113. The grating 103 / 113 generates a +-1 level light beam which is projected onto the object plane 124 after passing through several lenses to generate structured light fringes.

[0104] In Figure 4, gratings 103 and 113 are mounted orthogonally to each other, producing two mutually orthogonal interference fringes. The gratings' orientation angles can be 0° and 90°, 15° and 105°, and so on. In Figure 4, the solid line represents the light beam of the first illumination path, while the dashed line represents the light beam of the second illumination path. Gratings 103 / 113 are modulated gratings with high transmittance in the + / -1 level. The preferred gratings 103 / 113 have a total transmittance of >70% in the + / -1 level, improving the energy efficiency of the optical system compared to conventional gratings.

[0105] The grating 103 / 113 is mounted on a one-dimensional translation stage (not shown), which drives the grating to move back and forth to achieve different grating phase switching. The three different phases are Preferably, the one-dimensional translation stage is driven by piezoelectric ceramics to achieve fast movement. Preferably, the piezoelectric translation stage load grating should have a settling time of less than 10ms (milliseconds) when moving in 2um (micrometer) steps to meet the system's fast imaging requirements.

[0106] Lenses 104 / 114 and lenses 106 / 116 form a Fourier 4f system, with gratings 103 / 113 placed on the front focal plane of the 4f system, energy level filters 105 / 115 placed on the spectrum plane of the 4f system, and galvanometers 107 / 117 placed on the back focal plane of the 4f system. Energy level filters 104 / 114 only allow the + / - 1st order beams emitted from gratings 103 / 113 to pass through, filtering out beams of other energy levels to prevent interference with the structured light. Specifically, the energy level filter can be a spatial filter.

[0107] In Figure 4, 107 indicates that the galvanometer is at a first angle, and 117 indicates that the galvanometer is at a second angle. The angle between the two illumination light paths is designed according to the actual mechanical layout requirements. Generally speaking, the smaller the angle, the faster the galvanometer switching speed. Preferably, the angle between the two light paths can be set to 10°. The galvanometer 107 / 117 reflects the structured light of the two different illumination light paths onto the object surface by quickly switching the angle. The structured light of the two light paths has the same phase and the angles are orthogonal to each other. In order to meet the requirements of high-speed imaging, the setting time of the galvanometer 107 / 117 should be less than 20ms when switching back and forth at a mechanical angle of 10°. The preferred galvanometer system can achieve a setting time of less than 10ms. When the galvanometer is at the first angle 107, the solid line light beam of the first illumination light path is reflected into the object space; when the galvanometer rotates to the second angle 117, the dotted line light beam of the second illumination light path will be reflected into the object space.

[0108] Lens 121 and objective lens 123 form a second 4f system, with galvanometer mirrors 107 / 117 located on the front focal plane of the 4f system, and object plane 124 located on the back focal plane. The two front and rear 4f systems allow the + / -1 order beams generated by gratings 103 / 113 to overlap on the object plane, thereby producing the desired structured light fringes. To accommodate the different laser wavelengths of light sources 101 / 111, lenses 104 / 114, 106 / 116, and 121 should be a set of achromatic lenses; preferably, through appropriate optical design, lenses 104 / 114, 106 / 116, and 121 can be the same lens to reduce hardware costs.

[0109] The illumination light beam emitted from lens 121 toward the object is reflected by the dichroic mirror and reaches the object plane 124. The dichroic mirror 122 has a high reflectivity near wavelengths of 532nm and 660nm, and can reflect the illumination structured light onto the object plane 124. At the same time, it has a high transmittance for other visible light bands, allowing the fluorescence emitted from the object plane 124 to be transmitted to the camera 126.

[0110] The sample 124 is generally based on a silicon wafer or glass, on which a biological tissue slice with a fluorescent dye or a regularly arranged DNA (deoxyribonucleic acid) nanosphere is attached. Preferably, the DNA nanospheres are spaced 360nm apart. When irradiated by laser, they emit a fluorescent signal. The fluorescent signal is collected by the camera 126 after passing through the objective lens 123 and the tube lens 125. Depending on the type of sample to be tested and the type of fluorescent dye, the tube lens 125 and the camera 126 can be one channel or multiple channels. For example, in a common four-color imaging gene sequencing system, the tube lens 125 and the camera 126 will have four channels.

[0111] Please refer to Figures 5 and 6. Figure 5 is a front view of the structured light illumination system in this embodiment, and Figure 6 is an oblique view of the structured light illumination system in this embodiment. This structured light illumination system is used to generate the structured light required for illumination and includes a laser light source, a grating, a piezoelectric displacement stage, a galvanometer, and several lenses. In Figure 5, the left and right optical paths are arranged symmetrically, and the gratings of the two optical paths are installed at an angle of 90°. This produces two mutually orthogonal structured light stripes. At the same time, the rapid swinging of the galvanometer 221 can achieve high-speed switching of the direction of the structured light stripes.

[0112] In this embodiment, all components are installed with the base plate 200 as a reference plane, thereby ensuring that the optical axes are on the same plane.

[0113] In Figure 5, light sources 201 / 211 are laser sources, using optical fiber output with a numerical aperture of 0.22. The fiber port type is either FC / APC (a type of fiber port) or SMA (a type of fiber port). Light sources 201 / 211 are connected to the illumination system via the corresponding optical fiber interface. Light sources 201 / 211 each have two wavelengths: 532nm and 660nm, with a power of 4W at each wavelength.

[0114] The light beam emitted by light source 201 / 211 passes through collimating lenses 202 / 212 and is projected onto gratings 203 / 213. Collimating lenses 202 / 212 are achromatic lenses with a focal length of 50 mm. The first and second gratings 203, 213 are mounted orthogonally to each other. For example, the first grating 203 is mounted at a 15° angle, while the second grating 213 is mounted at a 105° angle. Gratings 203 / 213 are modulated gratings with a ±1st order diffraction efficiency of approximately 80% and a line pair count of 117 lp / mm.

[0115] The first grating 203 and the second grating 213 are mounted on the first piezoelectric displacement stage 204 and the second piezoelectric displacement stage 214 respectively. The piezoelectric displacement stages 204 / 214 load the gratings 203 / 213 to move back and forth to achieve switching between three different phases.

[0116] In this embodiment, lenses 205 / 215, 207 / 217, and 222 are identical, achromatic doublets with an effective focal length of 91 mm. Lenses 205 / 215 and 207 / 217 form the first 4f optical system. Gratings 203 / 213 are positioned at the front focal plane of the 4f optical system, galvanometer mirror 221 is positioned at the back focal plane, and energy level filters 206 / 216 are positioned at the relay plane of the 4f optical system to implement energy level filtering. Lenses 205 / 215, 207 / 217, and energy level filters 206 / 216 feature a mechanical structure with vertical fine-tuning to ensure the accuracy of the 4f system's focal plane.

[0117] As shown in Figure 7, the energy level filter 206 / 216 only allows the +-1 order light beams emitted by the grating 203 / 213 to pass through, and the light beams of other energy levels will be blocked. There is a mark point in the center of the energy level filter 206 / 216 for aligning the zero order light beam emitted by the grating 203 / 213.

[0118] Galvanometer 221 is a high-speed galvanometer capable of rapid, small-angle deflection. By varying the angle of galvanometer 221, either the left or right illumination beam can be selected to enter the object space. The left and right illumination beams have different structured light orientation angles, thereby switching the direction of the stripes. In this embodiment, galvanometer 221 takes less than 10ms to swing at a 10° angle.

[0119] The lens 222 and the objective lens (not shown) form a second 4f optical system, thereby imaging the structured light onto the object surface. The lens 222 has the function of front-back adjustment, which can adjust the focal plane of the structured light to ensure that it is imaged onto the sample surface.

[0120] This embodiment also proposes another structured light illumination system that can replace the above structured light illumination system. The key technical point of this structured light illumination system is that two illumination structured lights in different directions use light sources with different polarization states, and then use a polarization beam combiner (PBC) to combine them to improve the system's light efficiency.

[0121] As shown in Figure 8 , the vertical and horizontal illumination light paths have different structured light directions and polarization orientations. For example, the structured light direction of the vertical illumination light path is 0°, with s-polarization; the structured light direction of the horizontal illumination light path is 90°, with p-polarization. When the two illumination light beams pass through the polarization beam combiner 610, they both have a transmission efficiency exceeding 90%. The two light beams then pass through the focusing lens 621 and enter the object space. The subsequent optical path structure is the same as in the first embodiment.

[0122] In this embodiment, light sources 601 / 611 are laser light sources. Light sources 601 / 611 have two output wavelengths, 532 nm and 660 nm, with an output power of 4 W at each wavelength. Light sources 601 / 611 use spatial light output, with the polarization state of first light source 601 being p-polarization and the polarization state of second light source 611 being s-polarization.

[0123] The light beams emitted by the light sources 601 / 611 directly illuminate the gratings 602 / 612 to generate structured light in different directions. The polarization state of the first light source 601 is p-polarization, and the corresponding stripe direction is 90°; the polarization state of the second light source 611 is s-polarization, and the corresponding stripe direction is 0°.

[0124] Lenses 603 / 613 and lenses 605 / 615 form a 4f system. Gratings 602 / 612 are placed on the front focal plane of the 4f system. Energy level filters 604 / 614 are placed on the spectrum plane of the 4f system. Polarization beam combiner 610 is placed on the back focal plane of the 4f system. Energy level filters 604 / 614 filter the beam emitted by gratings 602 / 612, allowing only ±1 level to pass.

[0125] Polarization beam combiner 610 is a common polarization optical component. It has a transmittance of over 90% for horizontally incident p-polarized light and a reflectivity of over 90% for vertically incident s-polarized light. Using polarization beam combiner 610 instead of traditional semi-transparent and semi-reflective mirrors can greatly improve the system's optical efficiency.

[0126] Lens 621 and objective lens 623 form a second 4f system. Polarization beam combiner 610 is located at the front focal plane of the 4f system, and object plane 624 is located at the back focal plane. The two 4f systems allow the + / -1 order beams generated by gratings 602 / 612 to overlap on the object plane, thereby producing the desired structured light fringes. The illumination beam directed toward the object from lens 621 is reflected by dichroic mirror 622 and reaches object plane 624. Dichroic mirror 622 has high reflectivity near wavelengths of 532nm and 660nm, reflecting the illumination structured light onto object plane 624. It also has high transmittance for other visible light bands, allowing fluorescence emitted from object plane 624 to be transmitted to camera 626.

[0127] Sample 624 is typically a silicon wafer or glass substrate, with a biological tissue slice or regularly arranged DNA nanospheres coated with fluorescent dye attached to it. Preferably, the DNA nanospheres are spaced 360 nm apart. When irradiated by laser light, they emit a fluorescent signal, which is collected by camera 626 after passing through objective lens 623 and tube lens 625. Depending on the type of sample to be tested and the type of fluorescent dye, tube lens 625 and camera 626 can be single or multiple. For example, a common four-color imaging gene sequencing system would have four tube lenses 625 and camera 626.

[0128] Example 3

[0129] Please refer to FIG9 , which is a flow chart of the structured light illumination method in this embodiment. Specifically, as shown in FIG9 , the structured light illumination method is applied to the structured light illumination system in embodiment 1 or embodiment 2, and the structured light illumination method includes:

[0130] S101, controlling a translation stage to move a first grating of at least two gratings to a first preset position, causing a first light source of at least two light sources to emit light beams, which are guided by a beam merging and guiding unit to merge into an object space to generate structured light of a first phase in a first direction;

[0131] S102, controlling the translation stage to sequentially move the first grating to other preset positions, and the first light source emits light beams, which are guided by the beam merging and guiding unit and converged into the object space to generate structured light of other phases in the first direction;

[0132] S103, controlling the translation stage to sequentially move the other gratings of the at least two gratings to preset positions, and the other light sources of the at least two light sources emit light beams, which are guided by the light beam merging and guiding unit and merged into the object space to generate structured light in other directions.

[0133] The structured light illumination method of this embodiment is further described below by taking examples.

[0134] For the structured light illumination system using a high-speed galvanometer in Example 2, based on the principle of super-resolution imaging, six images must be collected for each field of view (FOV) of sample 124. To improve the system's frame rate, this example proposes a fast-response lower-computer control system. As shown in Figure 10, the lower-computer system is enclosed in the dotted box, and its logic sequence is as follows:

[0135] (1) The host computer issues a photo-taking command, the galvanometer rotates to the first angle 107, and at the same time the first translation stage (not shown) carries the first grating 103 and moves it to the first position, corresponding to the first direction and the first phase of the structured light;

[0136] (2) After completing action (1), the first laser 101 is turned on, and the camera 126 collects the first set of images. After the collection is completed, the first laser 101 is turned off;

[0137] (3) Similarly, the first grating 103 moves to the second and third positions, corresponding to the second phase in the first direction and the third phase in the first direction of the structured light, and then the camera 126 collects the second and third sets of images, respectively. At this point, the three sets of phase images in the first direction of the structured light are collected;

[0138] (4) The galvanometer mirror rotates to a second angle 117; the second translation stage (not shown) loads the second grating 113 and moves it to the first position;

[0139] (5) After completing action (4), the second laser 111 is turned on, and the camera 126 simultaneously collects the fourth set of images. After the collection is completed, the second laser 111 is turned off;

[0140] (6) Similarly, the second grating 113 moves to the second and third positions, corresponding to the second direction phase 2 and the second direction phase 3 of the structured light, and then the camera 126 collects the fifth and sixth groups of pictures, respectively, and turns off the second laser 111; at this point, all six groups of pictures are collected for the current FOV;

[0141] (7) End the current FOV image acquisition process, the lower computer sends a feedback signal to the upper computer, moves to the next FOV to take a picture, and repeats the above actions.

[0142] For the structured light illumination system using the polarization beam combiner in Example 2, the photographing sequence of the structured light illumination method of this embodiment is as follows:

[0143] 1) The first grating 602 moves to a first phase position, the first laser 601 is turned on, and the camera 626 captures a first set of images;

[0144] 2) The first grating 602 moves to the second and third phase positions respectively, and the camera 626 collects the second and third sets of images. After the collection is completed, the first laser 601 is turned off;

[0145] 3) The second grating 612 moves to the first phase position, the second laser 611 is turned on, and the camera 626 collects a fourth set of images;

[0146] 4) The second grating 612 moves to the second and third phase positions respectively, and the camera 626 collects the fifth and sixth groups of images. After the collection is completed, the second laser 611 is turned off.

[0147] 5) End the current FOV image acquisition process, the lower computer sends a feedback signal to the upper computer, moves to the next FOV to take a picture, and repeats the above steps.

[0148] Example 4

[0149] This embodiment provides a super-resolution optical system, which includes a substrate, an imaging module, an autofocus module, and an illumination module; the imaging module, the autofocus module, and the illumination module are fixed on the substrate; the illumination module adopts the structured light illumination system of embodiment 1 or embodiment 2;

[0150] The autofocus module is coupled to the lighting module via a galvanometer, and the autofocus module is used to drive the objective lens to achieve autofocus;

[0151] The lighting module is used to emit a lighting structure light beam which is projected onto the surface of the sample to be photographed through the galvanometer and the objective lens;

[0152] The imaging module is used to receive the optical signal generated by the sample to be photographed after being irradiated by the illumination structure light beam; the optical signal is divided into several optical signals with different wavelengths after passing through the objective lens and several dichroic mirrors.

[0153] The following examples further illustrate the principle structure of the super-resolution optical system and the specific implementation of the imaging module. This system, combined with the structured light illumination system described in Example 1 or Example 2, forms a complete super-resolution optical system. As shown in Figures 11-13, Figure 11 is a front view of the optical system structure, Figure 12 is a first oblique view of the optical system structure, and Figure 13 is a second oblique view of the optical system structure. To better illustrate the details of the optical system, some structural support components are hidden in the figures, but this does not affect the functionality of the entire optical system.

[0154] The optical system structure in Figures 11-13 includes a substrate 300, an imaging module 301, an illumination module 302, an autofocus module 303, and a sample 304. The entire optical system is based on substrate 300, on which imaging module 301, illumination module 302, and autofocus module 303 are mounted. The illumination module 302 has been described in detail in Example 1. In this example, the structure and function of imaging module 301 and autofocus module 303 will be described in detail using a four-color gene sequencing system as an example.

[0155] Figure 14 shows a schematic diagram of the optical path structure of the super-resolution optical system imaging module. After the illumination module 406 is coupled to the autofocus module 405 via the galvanometer mirror 404, the light enters the object space via the first dichroic mirror 403. In this embodiment, the galvanometer mirror 404 is also a dichroic mirror, specifically a 750nm long-pass dichroic mirror. The autofocus module 405 operates at a wavelength of 820nm.

[0156] The detection light emitted by the autofocus module 405 passes through the galvanometer 404, the first dichroic mirror 403, the objective lens 402, and finally reaches the sample 401. The light then returns to the autofocus module 405 along the original path. The autofocus module 405 calculates the defocus value of the sample 401 based on the returned detection signal and drives the objective lens 402 to focus, thus achieving the autofocus function.

[0157] In FIG14 , the illumination structured light beam emitted by the illumination module 406 is reflected by the galvanometer 404 and the first dichroic mirror 403 , and then projected onto the surface of the sample 401 through the objective lens 402 .

[0158] The fluorescence signal emitted by the sample 401 is collected by the objective lens 402 and then divided into four paths according to the fluorescence wavelength from short to long through several dichroic mirrors;

[0159] The first dichroic mirror 403 has a reflectivity of more than 90% near 532nm, 660nm and 820nm, and a transmittance of more than 90% for other visible light bands; the second dichroic mirror 407 has a spectral parameter of 660nm long pass; the third dichroic mirror 408 has a spectral parameter of 585nm long pass; the fourth dichroic mirror 409 has a spectral parameter of 705nm long pass;

[0160] From the above-mentioned dichroic mirror spectral parameters, it can be seen that the wavelength of the first fluorescent signal is 550nm-580nm, which is reflected by the second dichroic mirror 407, then passes through the third dichroic mirror 408, and finally is focused by the tube lens 1 411 to the camera 1 412;

[0161] The second fluorescent signal has a wavelength of 590nm-640nm, is reflected by the second dichroic mirror 407, then reflected by the third dichroic mirror 408, and finally focused by the second tube lens 421 to the second camera 422;

[0162] The third fluorescent signal has a wavelength of 680nm-695nm, passes through the second dichroic mirror 407, is reflected by the fourth dichroic mirror 409, and is finally focused by the third tube lens 431 to the third camera 432;

[0163] The fourth fluorescent signal has a wavelength of 710 nm to 770 nm, passes through a second dichroic mirror 407, then a fourth dichroic mirror 409, and finally is focused by a fourth tube lens 441 onto a fourth camera 442.

[0164] Figures 15 and 16 show the physical structure diagrams of the super-resolution optical system imaging module.

[0165] The imaging module is mounted on an imaging substrate 500 , and a plurality of dichroic mirrors, a reflector, a tube lens and a camera are all mounted on the substrate 500 .

[0166] The illumination module and the auto-focus module are connected to the imaging module through the port where the first dichroic mirror 503 is located, and the integrated structure is shown in Figures 11-13.

[0167] The fluorescence signal emitted by the sample 501 is collected by the objective lens 502 and then divided into four paths according to the fluorescence wavelength from short to long through several dichroic mirrors;

[0168] The dichroic mirrors 503 / 507 / 508 / 509 all have a structure for fine-tuning the angle to ensure the accuracy of the optical axis angle.

[0169] The tube lenses 511 / 521 / 531 / 541 collect the fluorescence signals emitted from the object side and focus them into the cameras 512 / 522 / 532 / 542 respectively. The focal length of the tube lenses 511 / 521 / 531 / 541 is 246 mm, and they are all achromatic lenses.

[0170] To meet the detection capability of a large field of view, the cameras 512 / 522 / 532 / 542 are CMOS cameras with a resolution of 7000*7000 pixels and a high quantum conversion efficiency (QE).

[0171] A reflecting mirror 513 / 523 / 533 / 543 is added between the tube lens 511 / 521 / 531 / 541 and the camera 512 / 522 / 532 / 542 to fold the light path and reduce the size of the optical module.

[0172] Example 5

[0173] Figure 17 is a schematic diagram of the structure of an electronic device provided in Example 5 of the present invention. The electronic device includes a memory, a processor, and a computer program stored in the memory and executable on the processor. When the processor executes the program, it implements the structured light illumination method of Example 3. The electronic device 30 shown in Figure 17 is merely an example and should not limit the functionality or scope of use of the embodiments of the present invention.

[0174] As shown in FIG17 , the electronic device 30 may be a general-purpose computing device, such as a server device. Components of the electronic device 30 may include, but are not limited to, the at least one processor 31, the at least one memory 32, and a bus 33 connecting various system components (including the memory 32 and the processor 31).

[0175] The bus 33 includes a data bus, an address bus, and a control bus.

[0176] The memory 32 may include a volatile memory, such as a random access memory (RAM) 321 and / or a cache memory 322 , and may further include a read-only memory (ROM) 323 .

[0177] The memory 32 may also include a program / utility 325 having a set (at least one) of program modules 324, such program modules 324 including but not limited to: an operating system, one or more application programs, other program modules, and program data, each of which or some combination may include an implementation of a network environment.

[0178] The processor 31 executes various functional applications and data processing by running the computer programs stored in the memory 32 , such as the structured light illumination method of embodiment 3 of the present invention.

[0179] The electronic device 30 can also communicate with one or more external devices 34 (e.g., a keyboard, pointing device, etc.). This communication can occur via an input / output (I / O) interface 35. Furthermore, the model-generating device 30 can also communicate with one or more networks (e.g., a local area network (LAN), a wide area network (WAN), and / or a public network, such as the Internet) via a network adapter 36. As shown, the network adapter 36 communicates with other modules of the model-generating device 30 via a bus 33. It should be understood that, although not shown, other hardware and / or software modules can be used in conjunction with the model-generating device 30, including but not limited to microcode, device drivers, redundant processors, external disk drive arrays, RAID (RAID) systems, tape drives, and data backup storage systems.

[0180] It should be noted that although several units / modules or sub-units / modules of the electronic device are mentioned in the detailed description above, this division is merely exemplary and not mandatory. In fact, according to embodiments of the present invention, the features and functions of two or more units / modules described above may be embodied in a single unit / module. Conversely, the features and functions of a single unit / module described above may be further divided and embodied by multiple units / modules.

[0181] Example 6

[0182] This embodiment provides a computer-readable storage medium having a computer program stored thereon. When the program is executed by a processor, the structured light illumination method of embodiment 3 is implemented.

[0183] Specifically, the readable storage medium may include but is not limited to: a portable disk, a hard disk, a random access memory, a read-only memory, an erasable programmable read-only memory, an optical storage device, a magnetic storage device, or any suitable combination thereof.

[0184] In a possible implementation manner, the present invention may also be implemented in the form of a program product, which includes program code. When the program product is run on a terminal device, the program code is used to enable the terminal device to execute the structured light illumination method of embodiment 3.

[0185] The program code for executing the present invention may be written in any combination of one or more programming languages, and may be executed entirely on the user device, partially on the user device, as an independent software package, partially on the user device and partially on a remote device, or entirely on the remote device.

[0186] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A structured light illumination system, characterized in that: The structured light illumination system comprises: At least two light sources, the light sources are used to generate light beams of preset wavelengths; At least two gratings, respectively located on the light paths of the at least two light sources, the gratings being used to generate interference fringes; A translation stage, the grating is arranged on the translation stage, and the translation stage is used to move the grating to perform phase switching; A light beam merging and guiding unit, wherein the light paths converge at the light beam merging and guiding unit, and the light beam merging and guiding unit is used to guide the light beams to merge into the object space.

2. The structured light illumination system according to claim 1, characterized in that: The light beam merging and guiding unit comprises: A galvanometer, the optical paths intersect at the galvanometer, and the galvanometer is used to reflect the light beams to the object space by swinging at at least two preset angles.

3. The structured light illumination system according to claim 1, wherein: The structured light illumination system comprises: A first light source and a second light source, wherein the first light source and the second light source are used to generate light beams with different polarization directions; The light beam merging and guiding unit comprises: A polarization beam combiner, wherein the optical paths intersect at the polarization beam combiner, and the polarization beam combiner is used to reflect the light beam of the first light source to the object space, and the light beam of the second light source passes through the polarization beam combiner to reach the object space.

4. The structured light illumination system according to claim 1, wherein: The at least two gratings are arranged in directions orthogonal to each other so as to generate mutually orthogonal interference fringes; and / or, The at least two gratings include a modulated grating having a high transmittance at a preselected energy level.

5. The structured light illumination system according to claim 1, wherein: The light source is specifically used to generate light beams of at least two different preset wavelengths; and / or, The structured light illumination system further comprises: At least two collimating lenses are respectively located on the optical path, and the light beam reaches the grating after being collimated by the collimating lenses.

6. The structured light illumination system according to claim 1, wherein: The structured light illumination system further comprises: At least two filter modules are respectively located on the optical path and are used to filter out light beams outside the preselected energy level so that the light beams with the preselected energy level can reach the light beam merging and guiding unit.

7. The structured light illumination system according to claim 6, wherein: The filtering module comprises: The first lens and the second lens are used to form a first 4f system; the grating is located on the front focal plane of the first 4f system, and the light beam merging and guiding unit is located on the back focal plane of the first 4f system; The energy level filter is located on the frequency spectrum plane of the first 4f system and is used to filter out the light beams outside the preselected energy level so that the light beams of the preselected energy level can reach the light beam merging and guiding unit.

8. The structured light illumination system according to any one of claims 1 to 7, characterized in that: The structured light illumination system further comprises: The third lens and the fourth lens are used to form a second 4f system; the light beam merging and guiding unit is located on the front focal plane of the second 4f system, and the object plane is located on the back focal plane of the second 4f system.

9. The structured light illumination system according to claim 8, characterized in that: The structured light illumination system further comprises: A dichroic mirror, located on the optical path between the third lens and the fourth lens, and used for reflecting the structured light emitted by the third lens toward the object; The object side signal generated by the structured light irradiation is received after passing through the dichroic mirror.

10. A structured light illumination method, characterized in that: The structured light illumination method is applied to the structured light illumination system according to any one of claims 1 to 9, and the structured light illumination method comprises: Controlling the translation stage to move a first grating of at least two gratings to a first preset position, and a first light source of at least two light sources emits a light beam, and after being guided by a light beam merging and guiding unit, the light beam is merged into an object space to generate a structured light of a first phase in a first direction; The translation stage is controlled to sequentially move the first grating to other preset positions, and the first light source emits a light beam, which is guided by the light beam merging and guiding unit, and then merged into the object space to generate structured light of other phases in the first direction; The displacement stage is controlled to sequentially move other gratings of the at least two gratings to preset positions, and other light sources of the at least two light sources emit light beams, which are guided by the light beam merging and guiding unit to merge into the object space to generate structured light in other directions.

11. A super-resolution optical system, characterized in that: The super-resolution optical system comprises a substrate, an imaging module, an auto-focus module and an illumination module; the imaging module, the auto-focus module and the illumination module are fixed on the substrate; the illumination module adopts the structured light illumination system according to any one of claims 1 to 9; The auto-focus module is coupled to the lighting module via a galvanometer, and the auto-focus module is used to drive the objective lens to achieve auto-focus; The lighting module is used to emit a lighting structure light beam which is projected onto the surface of the sample to be photographed through the galvanometer and the objective lens; The imaging module is used to receive the optical signal generated by the sample to be photographed after being irradiated by the illumination structure light beam; The optical signal is divided into a plurality of optical signals with different wavelengths after passing through an objective lens and a plurality of dichroic mirrors.

12. An electronic device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the structured light illumination method according to claim 10 is implemented.

13. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the structured light illumination method according to claim 10 is implemented.

Images