WO2023019400A1 - 显微成像装置及其光照芯片、成像方法、电子设备和介质 - Google Patents

显微成像装置及其光照芯片、成像方法、电子设备和介质 Download PDF

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WO2023019400A1
WO2023019400A1 PCT/CN2021/112816 CN2021112816W WO2023019400A1 WO 2023019400 A1 WO2023019400 A1 WO 2023019400A1 CN 2021112816 W CN2021112816 W CN 2021112816W WO 2023019400 A1 WO2023019400 A1 WO 2023019400A1
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Prior art keywords
illumination
light
lens
chip
microscopic imaging
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PCT/CN2021/112816
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English (en)
French (fr)
Inventor
周藩
杨少壮
倪洁蕾
沈梦哲
徐讯
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深圳华大生命科学研究院
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Priority to CN202180098302.5A priority Critical patent/CN117355784A/zh
Priority to PCT/CN2021/112816 priority patent/WO2023019400A1/zh
Publication of WO2023019400A1 publication Critical patent/WO2023019400A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens

Definitions

  • the present disclosure relates to the technical field of optical imaging, in particular to a super-resolution microscopic imaging device, an illumination chip, an imaging method, electronic equipment and media.
  • optical diffraction limit The resolution of traditional optical microscopy imaging systems is limited by the optical diffraction limit, which prevents the development of optical microscopy imaging technology.
  • the theory of optical diffraction limit was proposed by German scientist E. Abbe (name) in 1873.
  • the resolution of the optical system cannot exceed ⁇ /(2*NA), where ⁇ is the wavelength of the incident light, and NA is the numerical aperture of the optical system. This theory led scientists to believe for much of the 20th century that optical microscopy would never be able to see finer dimensions, such as the interactions of individual molecules inside cells.
  • the structured illumination microscopic imaging method Compared with other super-resolution methods, the structured illumination microscopic imaging method has the advantages of fast imaging speed, low requirement for dyes, simple optical path structure, small light damage, and can be used for real-time dynamic three-dimensional imaging of living cells, etc., and is widely used in biomedicine field.
  • the traditional SIM is based on the frequency domain solution, and the reconstruction of super-resolution images requires at least nine consecutive images.
  • the reconstruction method proposed by Professor Gustafsson three different phase values need to be obtained in each direction of illumination, and high-frequency information is obtained by solving linear equations, so three images need to be taken; in addition, in order to obtain super-resolution in each direction , the lighting direction needs to be rotated by 3 angles (generally 0°, 60°, 120°), that is to say, a total of 9 images are required.
  • 3 angles generally 0°, 60°, 120°
  • the reconstruction algorithm based on the frequency domain solution inevitably introduces artifacts, which confuses the judgment of the true shape of the observed sample structure.
  • the existing microscopic imaging methods generally have the following defects:
  • the main purpose of the present disclosure is to provide a microscopic imaging device and its light-emitting chip, imaging method, electronic equipment and medium, so as to improve the above-mentioned defects existing in the prior art.
  • an illumination chip for a microscopic imaging device including: an illumination array structure, including a substrate and a number of illumination units periodically distributed on the substrate; and an illumination well, It is arranged on a surface where the illumination unit extends on the illumination array structure, and the illumination well is divided into several placement units for placing samples, and each placement unit is respectively distributed on the corresponding illumination unit, wherein,
  • the illumination unit is used to generate surface plasmon structured light when illuminated by a light source to excite the fluorescent dye in the corresponding sample on the placement unit and generate a fluorescent signal.
  • the illumination chip further includes a substrate layer disposed on the substrate, the plurality of illumination units are respectively disposed in the substrate layer, and the illumination wells are disposed on the substrate layer. A surface on which the lighting unit extends.
  • the substrate layer includes: a first substrate layer configured to provide the plurality of illumination units; and a second substrate layer configured to be distributed between the extended upper surface of the illumination unit and the plurality of illumination units. Light wells contact between the lower surfaces of the substrate layers.
  • the material of the substrate layer includes silicon dioxide.
  • At least two placement units are distributed on both sides of the corresponding illumination unit.
  • the placing units are symmetrically distributed on the left or right side of the lighting unit
  • the illumination unit is used to generate the surface plasmon structured light to respectively excite the fluorescent dyes in the samples distributed on the placement units on the left and right sides of the illumination unit when they are respectively irradiated by light sources with symmetrical illumination angles. .
  • illumination units are periodically distributed on the substrate in the form of a regular polygon.
  • the illumination unit includes an illumination cylinder.
  • the illumination cylinder includes an illumination cylinder.
  • the material of the substrate includes a light-transmitting material; and/or, the material of the lighting unit includes a metal material; and/or, the material of the lighting well includes a light-impermeable material.
  • a microscopic imaging device comprising: the above-mentioned illumination chip for the microscopic imaging device; a light source configured to irradiate light to the illumination chip; a beam angle control device , configured to adjust the irradiation angle at which the light source irradiates light to the illumination chip; and an imaging processing device configured to generate at least two original images according to the fluorescence signals generated on the illumination chip at different illumination angles, And an overlay process is performed on at least two original images to generate a microscopic image.
  • the microscopic imaging device also includes a collimating lens, a reflector, a lens, a dichroic mirror, an objective lens, and a tube lens;
  • the collimating lens is used to receive light irradiated by the light source, and The collimated light is emitted to the reflector;
  • the reflector is used to reflect the light to the beam angle control device;
  • the beam angle control device is configured to receive the light from the reflector and emit The outgoing light at the first outgoing angle is sent to the lens;
  • the lens is used to irradiate the concentrated light to the dichroic mirror;
  • the dichroic mirror is used to reflect the received converged light to the objective lens;
  • the objective lens is used to irradiate the outgoing light at the third exit angle to the illumination chip to generate a fluorescence signal;
  • the objective lens is also used to collect the fluorescence signal generated on the illumination chip, and transmit the light through the dichroic A mirror emits to the tube lens;
  • the tube lens
  • the microscopic imaging device also includes a collimating lens, a first reflector, a second reflector, a first converging lens, a second converging lens, an objective lens, and a tube lens;
  • the collimating lens is used for receiving the light irradiated by the light source, and emitting the collimated light to the first reflector;
  • the first reflector is used to reflect the light to the beam angle control device;
  • the beam angle control device is configured to receive the light from the first reflective mirror, and emit the outgoing light at a first outgoing angle to the first converging lens;
  • the second reflective mirror is used for converging the first converging lens The converging light is reflected to the second converging lens;
  • the second converging lens is used to irradiate the outgoing light at the second exit angle onto the illumination chip;
  • the objective lens is used to collect the fluorescent signal generated on the illumination chip ;
  • the tube lens is used to converge the fluorescent signal collected by the objective lens to the imaging processing
  • the beam angle control device is configured to set the switching time of the irradiation angle to be less than 1 ms.
  • the beam angle control device includes a scanning galvanometer.
  • the light source includes any one or more of a laser light source, an LED (Light Emitting Diode) light source, and a mercury lamp.
  • a microscopic imaging method comprising: acquiring at least two original images, wherein the at least two original images are used for microscopic imaging under different illumination angles as described above generating the fluorescent signal generated on the illumination chip of the device; and performing superimposition processing on at least two original images to generate the microscopic image.
  • an electronic device including a memory, a processor, and a computer program stored in the memory and executable on the processor.
  • the processor executes the computer program, the above-mentioned display micro-imaging method.
  • a computer-readable medium having stored thereon computer instructions which, when executed by a processor, implement the microscopic imaging method as described above.
  • the positive progress effect of the present disclosure lies in: for arraying biological samples, by changing the illumination angle of the incident light, the selective excitation of the samples can be effectively achieved, and at least two images can be used to achieve at least 2 times the resolution improvement, thus effectively It greatly reduces the number of original images, does not require frequency domain reconstruction algorithms, produces less artifacts, does not affect sample observation, and effectively reduces the difficulty and cost of building a microscopic imaging device.
  • FIG. 1 is a schematic structural diagram of a microscopic imaging device according to an embodiment of the present disclosure.
  • Fig. 2 is a schematic cross-sectional structure diagram of an illumination chip used in a microscopic imaging device.
  • Fig. 3 is a schematic top view of the structure of the illumination array of the illumination chip used in the microscopic imaging device.
  • Fig. 4 is a schematic perspective view of the three-dimensional structure of the illumination array structure of the illumination chip used in the microscopic imaging device.
  • Fig. 5 is a schematic diagram of a three-dimensional structure of an illumination chip used in a microscopic imaging device.
  • Fig. 6 is a schematic diagram of the distribution of placement units for the light-emitting chip of the microscopic imaging device.
  • FIG. 7 is a schematic diagram showing the energy distribution of surface plasmon structured light generated by an illumination unit under an illumination angle of a light source.
  • Fig. 8 is a schematic diagram illustrating energy distribution of surface plasmon structured light generated by the illumination unit under another light source illumination angle.
  • Fig. 9 is a schematic diagram showing samples periodically distributed on the light-emitting chip used in the microscopic imaging device.
  • Fig. 10 is a schematic diagram showing the left sample of the illumination unit illuminated by surface plasmon structured light generated under an illumination angle of a light source.
  • Fig. 11 is a schematic diagram showing the surface plasmon structured light generated under another light source illumination angle to illuminate the sample on the right side of the illumination unit.
  • Fig. 12 is a schematic structural diagram of a microscopic imaging device according to another embodiment of the present disclosure.
  • FIG. 13 is a schematic flowchart of a microscopic imaging method according to another embodiment of the present disclosure.
  • Fig. 14 is a schematic structural diagram of an electronic device implementing a microscopic imaging method according to another embodiment of the present disclosure.
  • Light source 1 collimating lens 2;
  • Beam angle control device 4 first converging lens 5;
  • Light Unit 83 Light Well 84;
  • tube lens 11 imaging processing equipment 12;
  • lens 13 dichroic mirror 14;
  • Memory 32 RAM 321;
  • program module 324 program tool 325;
  • I/O interface 35 The I/O interface 35; network adapter 36.
  • references in the specification to "an embodiment,” “an alternative embodiment,” “another embodiment,” etc. indicate that the described embodiments may include a particular feature, structure, or characteristic, but each embodiment The specific feature, structure or characteristic may not necessarily be included. Moreover, such phrases are not necessarily referring to the same embodiment. Furthermore, when a particular feature, structure or characteristic is described in conjunction with an embodiment, it is within the purview of those skilled in the relevant arts to implement such feature, structure or characteristic in conjunction with other embodiments, whether or not explicitly described.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features.
  • plural means two or more.
  • the term “comprise” and any variations thereof, are intended to cover a non-exclusive inclusion.
  • connection should be interpreted in a broad sense, for example, it can be a fixed connection or a flexible connection.
  • Detachable connection, or integral connection it can be mechanical connection or electrical connection; it can be direct connection or indirect connection through an intermediary, and it can be the internal communication of two components.
  • the present embodiment provides a microscopic imaging device, including: an illumination chip for the microscopic imaging device; a light source configured to irradiate light to the illumination chip; a light source adjustment mechanism configured to adjust The irradiation angle at which the light source irradiates light to the illumination chip; the imaging processing device is configured to generate at least two original images according to the fluorescence signals generated on the illumination chip at different illumination angles, and perform superposition processing on the at least two original images to generate a display image. micro image.
  • the illumination chip used for the microscopic imaging device includes: an illumination array structure, including a substrate and a number of illumination units periodically distributed on the substrate; an illumination well, arranged on a surface where the illumination unit extends on the illumination array structure, and on the illumination well There are several placement units for placing samples, and each placement unit is distributed on the corresponding illumination unit, wherein the illumination unit is used to generate surface plasmon structured light to excite the samples on the corresponding placement unit when illuminated by the light source. fluorescent dyes and generate fluorescent signals.
  • the periodically distributed samples may be, for example, DNA (deoxyribonucleic acid) beads, quantum dots, fluorescent nano beads, etc. in the sequencer.
  • DNA deoxyribonucleic acid
  • quantum dots quantum dots
  • fluorescent nano beads etc. in the sequencer.
  • this embodiment does not specifically limit the type of samples, and corresponding settings and adjustments can be made according to actual needs, actual scenarios, or possible needs and scenarios.
  • the selective excitation of the samples is effectively achieved, and at least two images are needed to achieve at least a 2-fold increase in resolution, thereby effectively reducing the
  • the number of original images is reduced, no frequency domain reconstruction algorithm is required, less artifacts are generated, sample observation is not affected, and the difficulty and production cost of the microscopic imaging device are effectively reduced.
  • the microscopic imaging device mainly includes but is not limited to a light source 1, a collimator lens 2, a first reflector 3, a beam angle control device 4, First converging lens 5, second reflecting mirror 6, second converging lens 7, light chip 8 for micro imaging device, objective lens 10, tube lens 11, imaging processing equipment 12 and computer (not shown in the figure).
  • the light source 1 is configured to irradiate light to the illumination chip 8;
  • the collimator lens 2 is used to receive the light irradiated by the light source 1, and emit the collimated parallel light to the first reflector 3;
  • the first reflector 3 is used to direct the light Reflected to the beam angle control device 4;
  • the beam angle control device 4 is configured to receive light from the first reflector 3, and emit the exit light of the first exit angle to the first converging lens 5;
  • the second reflector 6 is used to pass through
  • the converging light converged by the first converging lens 5 is reflected to the second converging lens 7;
  • the second converging lens 7 is used to irradiate the outgoing light at the second outgoing angle (the angle with the normal line of the incident surface, for example, 60 degrees) to the light emitting chip 8, surface plasmon structured light is generated to excite the fluorescent dye in the sample 9 and generate a fluorescent signal;
  • the objective lens 10 is used to collect the fluorescent signal generated on
  • the imaging processing device 12 is connected in communication with the computer, and the imaging processing device 12 is configured to generate at least two original images according to the fluorescence signals generated on the illumination chip 8 at different illumination angles (for example, 60 degrees and -60 degrees as a symmetrical angle). , and perform an overlay process on at least two original images to generate a super-resolution microscopy image.
  • the beam angle control device 4 is configured to set the switching time of the irradiation angle to less than 1 ms, so that the switching time is much shorter than the time required for displacement and rotary platform movement in the traditional structured light microscopy imaging method .
  • the beam angle control device 4 can use the rotary motion platform to drive its mirror to rotate to realize the control of the light irradiation angle.
  • the beam angle control device 4 includes a scanning galvanometer, preferably a single-axis scanning galvanometer, more specifically XY scanning galvanometers may be included, but the type of beam angle control device 4 is not specifically limited. As long as the corresponding functions can be realized, corresponding settings and adjustments can be made according to actual needs, actual scenes, or possible needs and scenes.
  • the microscopic imaging device only needs to change the illumination angle of the incident light through the scanning galvanometer, and does not need to rotate and translate the grating, thus effectively reducing the construction difficulty and manufacturing cost of the microscopic imaging device.
  • the light source 1 includes any one or more of a laser light source, an LED light source, and a mercury lamp, but the type of light source 1 is not specifically limited, as long as the corresponding functions can be realized, the Or the needs and scenarios that may arise are set and adjusted accordingly.
  • the light chip 8 (also called a nano-chip) mainly includes a light array structure (also called a nano-array structure) and a light well 84 (also called a nano-well).
  • the illumination array structure mainly includes a substrate 81, a substrate layer (the material of the substrate layer includes silicon dioxide) and several illumination units 83, the substrate layer is arranged on the substrate 81, and several illumination units 83 and Regular hexagons (or other shapes such as regular quadrilaterals) are periodically arranged in the substrate layer and distributed on the substrate 81 , and the light wells 84 are arranged on a surface where the light unit 83 extends on the substrate layer.
  • the substrate layer mainly includes a first substrate layer 821 and a second substrate layer 822 (the dotted line in FIG. Constructed from exactly the same material).
  • the first substrate layer 821 is configured to arrange several illumination units 83, and the height of the first substrate layer 821 corresponds to the height of the illumination units 83, for example, 80 nm;
  • the second substrate layer 822 is configured to be distributed on the upper surface where the illumination units 83 extend Between the lower surface of the substrate layer in contact with the illumination well 84, the second substrate layer 822 can protect the illumination unit 83 and effectively prevent corrosion. Therefore, the height of the second substrate layer 822 can be adjusted according to actual needs. And set, for example, 20nm.
  • the illumination unit 83 can be an illumination cylinder, preferably an illumination cylinder, and the illumination unit 83 can be a solid metal, preferably silver or copper. , aluminum, gold, etc.
  • this embodiment does not specifically limit the shape and material of the lighting unit 83 , as long as the corresponding functions can be realized, corresponding settings and adjustments can be made according to actual requirements, actual scenarios, or possible requirements and scenarios.
  • the illumination well 84 is divided into several placement units 841 for placing the sample 9, and each placement unit 841 is respectively distributed on the corresponding illumination unit 83, wherein the illumination unit 83 is used to be irradiated by the light source. , surface plasmon structured light is generated to excite the fluorescent dye in the sample 9 on the corresponding placement unit 841 and generate a fluorescent signal.
  • the placement units are symmetrically distributed on the left or right side of the illumination unit 83, wherein the illumination unit 83 is used to be illuminated by light sources with symmetrical illumination angles.
  • the surface plasmon structured light is generated to respectively excite the fluorescent dyes in the samples distributed on the placement unit 841 on the left and right sides of the illumination unit 83 .
  • the illumination chip of this embodiment has a special structural design for periodically distributed samples and surface plasmon illumination characteristics.
  • the material of the substrate includes light-transmitting materials (for example, materials with high transmittance such as quartz and BK7), so that the light source passes through the substrate and irradiates the illumination unit.
  • the light well structure can be designed strictly according to the light array structure. The length and width parameters of the placement unit of the light well are determined according to the spacing between the light units, and the height of the light well is determined according to the sample size.
  • the material of the light well includes opaque materials, such as TiN (titanium nitride), to ensure that the plasma structured light generated when the incident light angle changes will not excite the partition wall sample.
  • the illumination unit selects different materials and size parameters according to the laser wavelength and resolution requirements, combined with finite element analysis and simulation.
  • the material of the illumination unit is silver, its diameter is 60nm, and its height is 60nm.
  • Pitch refers to the distance between the centers of two adjacent illumination units (i.e. illumination cylinders)
  • 150nm the surface plasmon has better resonance energy and meets the requirements of resolution improvement.
  • the illumination unit is shown with a white circle body, and its inner colored circle body shows the excitation degree (the closer to the internal excitation degree stronger), the illumination angles are +60 degrees (corresponding to Figure 7) and -60 degrees (corresponding to Figure 8), and the surface plasmons generated by the two symmetrical angles are symmetrically distributed on the left and right sides of the illumination unit.
  • the upper layer is designed light well.
  • Figure 9 shows the samples on the surface of the illuminated chip when the laser is not irradiated.
  • the unexcited samples in the figure are circled in black.
  • the surface plasmon generated excites the fluorescent dye in the sample, and the irradiation angle changes symmetrically.
  • the samples distributed on the left and right sides of the illumination unit are excited sequentially.
  • Figure 10 shows that the samples on the left side of the illumination unit are excited (considering the arrangement of the illumination units in Figure 6), and the excited samples are shown in gray circles.
  • Figure 11 shows the illumination The sample on the right side of the unit is excited (considered in conjunction with the arrangement of the illumination unit in Figure 6), and the excited sample is also shown in a gray circle.
  • the excitation light is irradiated to the beam angle control device through the quasi-lens and the first reflector, and the laser is emitted at a certain angle, as shown by the dotted line in Figure 1.
  • the laser excites the light array structure to generate surface plasmon structured light, as shown in Figure 7, the plasma is confined to the left side of the light unit, and the light well
  • the structure is designed according to the distribution of the illumination array structure, and every two placement units are distributed on the left and right sides of the illumination unit, as shown in Figure 6, so the plasma structured light only excites the samples in the placement unit on the left above the illumination unit, and the irradiated samples generate fluorescence Signal, as shown in Figure 10 (white indicates the generation of fluorescent signal), the objective lens collects the fluorescent signal generated by the sample, focuses on the
  • the beam angle control device Control the beam angle control device to change the angle of the outgoing light, which is symmetrical to the angle in step A.
  • the excitation light is finally irradiated on the illumination chip at a certain angle (for example -60 degrees), and the laser excitation light
  • the array structure generates surface plasmon structured light, as shown in Figure 8, the plasma is confined to the right side of the illumination unit, and the sample placed in the unit on the right side above the illumination unit is excited, and the sample on the right side is excited with a fluorescent signal, as shown in Figure 11 , the objective lens collects the fluorescent signal generated by the sample, and focuses on the imaging processing device through the tube lens to form the second original image.
  • the specific angle at which the light chip is finally irradiated in step A needs to be determined according to the time-domain finite element analysis simulation, which is related to the size, material, incident wavelength and other parameters of the light array structure.
  • the time-domain finite element analysis simulation which is related to the size, material, incident wavelength and other parameters of the light array structure.
  • the illumination unit laying on the substrate Silver cylinders with a height of 60nm and a diameter of 60nm (that is, the illumination unit), and when the illumination unit is periodically distributed in a regular hexagon, it is recommended to use angles of +60 degrees and -60 degrees according to the simulation results of time-domain finite element analysis.
  • the beam angle control device’s exit light angle is based on the magnification of the lens group composed of the first converging lens and the second converging lens, and combined with the angle at the light chip to calculate the angle that needs to be controlled.
  • the magnification of the lens group is 2 times
  • the irradiation angle of the light chip is +60 degrees and -60 degrees
  • the exit light angle of the beam angle control device is ⁇ 60 degrees/2, that is, +30 degrees and -30 degrees.
  • the optical path of the microscopic imaging device is simple, which reduces the difficulty of construction, is compatible with commonly used microscopic imaging systems, and reduces the production cost;
  • the output microscopic super-resolution image can theoretically achieve at least a 2-fold increase in resolution.
  • the microscopic imaging device mainly includes a light source 1, a collimating lens 2, a reflector 3', a beam angle control device 4, a lens 13, and a dichroic mirror 14.
  • Objective lens 10 tube lens 11, imaging processing device 12 and computer (not shown in the figure).
  • the structures and functions of the light source 1, the collimating lens 2, the reflector 3', the beam angle control device 4, the tube mirror 11, the imaging processing device 12 and the computer can refer to the corresponding components in the above-mentioned embodiment, so they will not be described again. A repeat.
  • the illumination light path is mainly replaced from the projection type as described above to the reflection type.
  • the collimating lens 2 is used to receive the light irradiated by the light source 1, and emit the collimated light to the reflector 3'; the reflector 3' is used to reflect the light to the beam angle control device 4; the beam angle control device 4 is configured to receive light from the mirror 3', and emit the outgoing light at the first outgoing angle to the lens 5; the lens 5 is used to irradiate the concentrated light to the dichroic mirror 14; the dichroic mirror 14 is used to reflect the received converging light to the objective lens 10; the objective lens 10 is used to irradiate the outgoing light at the third exit angle onto the illumination chip 8 to generate surface plasmon structured light to excite the sample 9 in the illumination well to emit fluorescence signals
  • the objective lens 10 is also used to collect the fluorescent signal generated on the illumination chip 8, and transmits to the tube lens 11 through the dichroic mirror 14; the tube lens 11 is
  • the microscopic imaging device provided in this embodiment is aimed at arranging biological samples in an array.
  • the sample By changing the illumination angle of the incident light, the sample can be selectively excited, and at least two images can be used to achieve at least a 2-fold increase in resolution, thereby The number of original images is effectively reduced, no frequency domain reconstruction algorithm is required, less artifacts are generated, sample observation is not affected, and the construction difficulty and production cost of the microscopic imaging device are effectively reduced.
  • the microscopic imaging device of the present disclosure is not limited to the second-generation gene sequencing system. If it is applied to the second-generation gene sequencing system, it can improve the sequencing throughput, reduce photobleaching and light loss, which is also important for sequencing, and reduce artifacts , improve the DNB (DNA nanoball) recognition rate, thereby increasing the Q30 (sequencing data) data value.
  • this embodiment provides a microscopic imaging method, which mainly includes the following steps:
  • Step 201 generating an irradiation angle adjustment signal to adjust the irradiation angle
  • Step 202 acquiring at least two original images by illuminating the fluorescent signals generated on the chip under different illumination angles
  • Step 203 Perform superposition processing on at least two original images to generate a microscopic image.
  • step 201 an illumination angle adjustment signal for the light source adjustment mechanism of the above-mentioned microscopic imaging device is generated and output to the light source adjustment mechanism, so as to adjust the illumination angle at which the light source irradiates light to the light chip.
  • step 202 at least two original images are acquired, wherein the at least two original images are generated by the fluorescence signals generated on the above-mentioned illumination chip used in the microscopic imaging device under different illumination angles.
  • the existing image superposition processing method may be used to perform superposition processing on at least two original images to generate a super-resolution microscopic image, which will not be described in detail one by one.
  • the microscopic imaging method provided in this embodiment can achieve super-resolution microscopic imaging by acquiring at least two original images, which improves the real-time performance and throughput of imaging, and effectively reduces dye photodrifting and sample light loss; using Spatial domain reconstruction, non-frequency domain reconstruction, reducing artifacts.
  • Fig. 14 is a schematic structural diagram of an electronic device provided according to this embodiment.
  • 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, the microscopic imaging method in the above embodiment is implemented.
  • the electronic device 30 shown in FIG. 14 is only an example, and should not limit the functions and scope of use of the embodiments of the present disclosure.
  • the electronic device 30 may be in the form of a general-purpose computing device, for example, it may be a server device.
  • Components of the electronic device 30 may include, but are not limited to: at least one processor 31 , at least one memory 32 , and a bus 33 connecting different system components (including the memory 32 and the processor 31 ).
  • the bus 33 includes a data bus, an address bus, and a control bus.
  • 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 .
  • RAM random access memory
  • ROM read only memory
  • Memory 32 may also include a program/utility tool 325 having a set (at least one) of program modules 324 including, but not limited to, an operating system, one or more application programs, other program modules, and program data, which Each or some combination of the examples may include the implementation of a network environment.
  • program/utility tool 325 having a set (at least one) of program modules 324 including, but not limited to, an operating system, one or more application programs, other program modules, and program data, which Each or some combination of the examples may include the implementation of a network environment.
  • the processor 31 executes various functional applications and data processing by executing the computer program stored in the memory 32 , such as the microscopic imaging method in the above embodiments of the present disclosure.
  • Electronic device 30 may also communicate with one or more external devices 34 (eg, keyboards, pointing devices, etc.). Such communication may occur through input/output (I/O) interface 35 .
  • the model generation device 30 can also communicate with one or more networks (eg, a local area network (LAN), a wide area network (WAN) and/or a public network, such as the Internet) via a network adapter 36 .
  • network adapter 36 communicates with other modules of model generation device 30 via bus 33 .
  • model generating device 30 may 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 (disk array) systems, tape drives, and data backup storage systems.
  • This embodiment also provides a computer-readable storage medium, on which a computer program is stored, and when the program is executed by a processor, the steps in the microscopic imaging method in the above embodiment are implemented.
  • the readable storage medium may more specifically include but not limited to: portable disk, hard disk, random access memory, read-only memory, erasable programmable read-only memory, optical storage device, magnetic storage device or any of the above-mentioned the right combination.
  • the present disclosure may also be implemented in the form of a program product, which includes program codes, and when the program product is executed on the terminal device, the program code is used to make the terminal device perform the display in the above embodiment. Steps in the microimaging method.
  • program code for executing the present disclosure may be written in any combination of one or more programming languages, and the program code may be completely executed on the user equipment, partially executed on the user equipment, or used as an independent software Package execution, partly on the user device and partly on the remote device, or entirely on the remote device.

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Abstract

一种显微成像装置及其光照芯片(8)、成像方法、电子设备(30)和介质。光照芯片(8)包括：光照阵列结构，包括衬底(81)及周期性分布于衬底(81)的若干光照单元(83)；光照井(84)，设置于光照阵列结构上光照单元(83)延伸的一表面，光照井(84)上划分有用于放置样品(9)的若干放置单元(841)，每个放置单元(841)分别分布于对应的光照单元(83)上，其中，光照单元(83)用于被光源(1)照射时，产生表面等离子体结构光以激发对应的放置单元(841)上的样品(9)中的荧光染料并产生荧光信号。这种显微成像装置及其光照芯片(8)、成像方法针对阵列排列生物样品（9），通过改变入射光照明角度，有效地实现样品（9）选择性被激发，可有效地实现分辨率的提升，从而有效地降低了原始图像数量。

Description

显微成像装置及其光照芯片、成像方法、电子设备和介质 技术领域
本公开涉及光学成像技术领域，具体涉及一种超分辨率显微成像装置及其光照芯片、成像方法、电子设备和介质。
背景技术
传统光学显微成像系统分辨率受光学衍射极限限制，阻止光学显微成像技术的发展。光学衍射极限理论由德国科学家E.Abbe(人名)在1873年提出，光学系统分辨率不能超过λ/(2*NA)，其中λ为入射光波长，NA为光学系统的数值孔径。该理论导致在20世纪大部分时间内，科学家们相信光学显微成像技术永远无法观察到更细微的尺寸，比如细胞内部单个分子的相互作用。
然而，20世纪末衍射极限理论被打破了。目前国际上已有多种成熟的超分辨显微成像方法：结构光照明显微成像方法(SIM),受激发射损耗显微成像方法(STED),随机光学重构显微成像方法(STORM)。其中，结构光照明显微成像方法由Gustafsson(人名)教授2000年提出，是在宽场显微成像基础上，使用强度周期调制的结构光照明样品，通过特定算法提取高频信息，重构出超分辨图像，实现衍射极限突破。相比于其他超分辨方法，结构光照明显微成像方法具有成像速度快、对染料要求低、光路结构简单、光损伤小，可用于活细胞实时动态三维成像等优点，被广泛应用于生物医学领域。
传统SIM基于频域求解，重构超分辨图像至少需要连续拍摄9张图像。根据Gustafsson教授提出的重建方法，在每个照明方向上需要获取3个不同相位值，通过解线性方程组获得高频信息，因此需要拍3张图像；此外，为了在每个方向上获得超分辨，照明方向需要旋转3个角度(一般0°，60°，120°)，也就是说总共需要9张图像。有时，为了获得更精细的重构数据，需要采集更多数量的图像。此外，基于频域求解的重构算法不可避免的引入伪影，混淆对观测样品结构的真实形态判断。
综上所述，现有的显微成像方法一般存在以下缺陷：
1)需要拍摄至少9张图像，影响拍照的实时性，并且导致系统通量低；
2)通过频域算法重构，不可避免会引入伪影，影响样品观测；
3)需要在不同相位不同旋转方向上连续采集多张图像，对结构光相位和旋转角度的精度要求高，从而对控制光栅的机械件的定位精度要求高，造成系统搭建难度和成本提高。
发明内容
本公开的主要目的在于，提供一种显微成像装置及其光照芯片、成像方法、电子设备和介质，以改善现有技术中存在的上述缺陷。
本公开是通过下述技术方案来解决上述技术问题：
根据本公开内容的一个方面，提供了一种用于显微成像装置的光照芯片，包括：光照阵列结构，包括衬底及周期性分布于所述衬底的若干光照单元；以及，光照井，设置于所述光照阵列结构上所述光照单元延伸的一表面，所述光照井上划分有用于放置样品的若干放置单元，每个所述放置单元分别分布于对应的所述光照单元上，其中，所述光照单元用于被光源照射时，产生表面等离子体结构光以激发对应的所述放置单元上的样品中的荧光染料并产生荧光信号。
作为可选实施方式，所述光照芯片还包括设置于所述衬底上的基板层，所述若干光照单元分别设置于所述基板层内，所述光照井设置于所述基板层上所述光照单元延伸的一表面。
作为可选实施方式，所述基板层包括：第一基板层，被配置为设置所述若干光照单元；以及，第二基板层，被配置为分布于所述光照单元延伸的上表面与所述光照井接触所述基板层的下表面之间。
作为可选实施方式，所述基板层的材料包括二氧化硅。
作为可选实施方式，至少每两个所述放置单元分布于对应的所述光照单元上的两侧。
作为可选实施方式，所述放置单元以对称方式分布于所述光照单元的左侧或右侧，
其中，所述光照单元用于分别被对称照射角度的光源照射时，产生所述表面等离子体结构光以分别激发分布于所述光照单元左右两侧的所述放置单元上的样品中的荧光染料。
作为可选实施方式，若干所述光照单元以正多边形方式周期性分布于所述衬底。
作为可选实施方式，所述光照单元包括光照柱体。
作为可选实施方式，所述光照柱体包括光照圆柱体。
作为可选实施方式，所述衬底的材料包括透光材料；和/或，所述光照单元的材料包括金属材料；和/或，所述光照井的材料包括不透光材料。
根据本公开内容的另一个方面，提供了一种显微成像装置，包括：如上述的用于显微成像装置的光照芯片；光源，被配置为向所述光照芯片照射光线；光束角度控制器件，被配置为调节所述光源向所述光照芯片照射光线的照射角度；以及，成像处理设备，被 配置为根据在不同照射角度下所述光照芯片上产生的荧光信号生成至少两张原始图像，并且对至少两张原始图像执行叠加处理以生成显微图像。
作为可选实施方式，所述显微成像装置还包括准直透镜、反射镜、透镜、二向色镜、物镜及筒镜；所述准直透镜用于接收所述光源照射的光线，并将准直后的光线发射至所述反射镜；所述反射镜用于将光线反射至所述光束角度控制器件；所述光束角度控制器件被配置为从所述反射镜接收所述光线，并且发射第一出射角度的出射光至所述透镜；所述透镜用于将会聚光照射至所述二向色镜；所述二向色镜用于将接收到的会聚光反射到所述物镜；所述物镜用于将第三出射角度的出射光照射至所述光照芯片以产生荧光信号；所述物镜还用于收集所述光照芯片上产生的所述荧光信号，并透过所述二向色镜发射到所述筒镜；所述筒镜用于将接收到的所述荧光信号会聚到所述成像处理设备。
作为可选实施方式，所述显微成像装置还包括准直透镜、第一反射镜、第二反射镜、第一会聚透镜、第二会聚透镜、物镜及筒镜；所述准直透镜用于接收所述光源照射的光线，并将准直后的光线发射至所述第一反射镜；所述第一反射镜用于将光线反射至所述光束角度控制器件；所述光束角度控制器件被配置为从所述第一反射镜接收所述光线，并且发射第一出射角度的出射光至所述第一会聚透镜；所述第二反射镜用于将经过所述第一会聚透镜会聚后的会聚光反射至所述第二会聚透镜；所述第二会聚透镜用于将第二出射角度的出射光照射至所述光照芯片上；所述物镜用于收集所述光照芯片上产生的荧光信号；所述筒镜用于将所述物镜收集到的所述荧光信号会聚到所述成像处理设备。
作为可选实施方式，所述光束角度控制器件被配置为将照射角度的切换时间设定为1ms以下。
作为可选实施方式，所述光束角度控制器件包括扫描振镜。
作为可选实施方式，所述光源包括激光光源、LED(发光二极管)光源及汞灯中的任意一种或多种。
根据本公开内容的另一个方面，提供了一种显微成像方法，包括：获取至少两张原始图像，其中，所述至少两张原始图像通过在不同照射角度下如上述的用于显微成像装置的光照芯片上产生的荧光信号来生成；以及，对至少两张原始图像执行叠加处理以生成显微图像。
根据本公开内容的另一个方面，提供了一种电子设备，包括存储器、处理器及存储在存储器上并可在处理器上执行的计算机程序，所述处理器执行计算机程序时实现如上述的显微成像方法。
根据本公开内容的另一个方面，提供了一种计算机可读介质，其上存储有计算机指 令，所述计算机指令在由处理器执行时实现如上述的显微成像方法。
在符合本领域常识的基础上，所述各可选条件，可任意组合，即得本公开各较佳实施例。
根据本公开内容，本领域技术人员可以理解本公开内容的其它方面。
本公开内容的积极进步效果在于：针对阵列排列生物样品，通过改变入射光照明角度，有效地实现样品选择性被激发，只需至少两幅图即可实现至少2倍分辨率的提升，从而有效地降低了原始图像数量，不需要频域重构算法，较少伪影的产生，不影响样品观测，而且有效地降低了显微成像装置的搭建难度和制作成本。
附图说明
在结合以下附图阅读本公开的实施例的详细描述之后，能够更好地理解本公开的所述特征和优点。在附图中，各组件不一定是按比例绘制，并且具有类似的相关特性或特征的组件可能具有相同或相近的附图标记。
图1为根据本公开内容的一实施例的显微成像装置的结构示意图。
图2为用于显微成像装置的光照芯片的剖面结构示意图。
图3为用于显微成像装置的光照芯片的光照阵列结构的俯视示意图。
图4为用于显微成像装置的光照芯片的光照阵列结构的立体结构示意图。
图5为用于显微成像装置的光照芯片的立体结构示意图。
图6为用于显微成像装置的光照芯片的放置单元的分布示意图。
图7为示出一光源照射角度下光照单元产生表面等离子体结构光能量分布的示意图。
图8为示出另一光源照射角度下光照单元产生表面等离子体结构光能量分布的示意图。
图9为示出样品周期性分布于用于显微成像装置的光照芯片上的样品示意图。
图10为示出一光源照射角度下产生的表面等离子体结构光照明光照单元左侧样品的示意图。
图11为示出另一光源照射角度下产生的表面等离子体结构光照明光照单元右侧样品的示意图。
图12为根据本公开内容的另一实施例的显微成像装置的结构示意图。
图13为根据本公开内容的另一实施例的显微成像方法的流程示意图。
图14为根据本公开内容的另一实施例的实现显微成像方法的电子设备的结构示意图。
附图标记说明：
光源                 1；准直透镜                   2；
第一反射镜           3；反射镜                     3’；
光束角度控制器件     4；第一会聚透镜               5；
第二反射镜           6；第二会聚透镜               7；
光照芯片             8；衬底                       81；
第一基板层           821；第二基板层               822；
光照单元             83；光照井                    84；
放置单元             841；
样品                 9；物镜                       10；
筒镜                 11；成像处理设备              12；
透镜                 13；二向色镜                  14；
电子设备             30；处理器                    31；
存储器               32；RAM                       321；
高速缓存存储器       322；ROM                      323；
程序模块             324；程序工具                 325；
总线                 33；外部设备                  34；
I/O接口              35；网络适配器                36。
具体实施方式
下面通过实施例的方式进一步说明本公开内容，但并不因此将本公开内容限制在所述的实施例范围之中。
应当注意，在说明书中对“一实施例”、“可选实施例”、“另一实施例”等的引用指示所描述的实施例可以包括特定的特征、结构或特性，但是每个实施例可能不一定包括该特定的特征、结构或特性。而且，这样的短语不一定指代相同的实施例。此外，当结合实施例描述特定特征、结构或特性时，无论是否被明确描述，结合其它实施例来实现这样的特征、结构或特性都在相关领域的技术人员的知识范围内。
在本公开内容的描述中，需要理解的是，术语“中心”、“横向”、“上”、“下”、“左”、“右”、“竖直”、“水平”、“顶”、“底”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本公开内容和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对 本公开内容的限制。此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本公开内容的描述中，除非另有说明，“多个”的含义是两个或两个以上。另外，术语“包括”及其任何变形，意图在于覆盖不排他的包含。
在本公开内容的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本公开内容中的具体含义。
这里所使用的术语仅仅是为了描述具体实施例而不意图限制示例性实施例。除非上下文明确地另有所指，否则这里所使用的单数形式“一个”、“一项”还意图包括复数。还应当理解的是，这里所使用的术语“包括”和/或“包含”规定所陈述的特征、整数、步骤、操作、单元和/或组件的存在，而不排除存在或添加一个或更多其他特征、整数、步骤、操作、单元、组件和/或其组合。
为了克服目前存在的上述缺陷，本实施例提供一种显微成像装置，包括：用于显微成像装置的光照芯片；光源，被配置为向光照芯片照射光线；光源调节机构，被配置为调节光源向光照芯片照射光线的照射角度；成像处理设备，被配置为根据在不同照射角度下光照芯片上产生的荧光信号生成至少两张原始图像，并且对至少两张原始图像执行叠加处理以生成显微图像。
其中，用于显微成像装置的光照芯片包括：光照阵列结构，包括衬底及周期性分布于衬底的若干光照单元；光照井，设置于光照阵列结构上光照单元延伸的一表面，光照井上划分有用于放置样品的若干放置单元，每个放置单元分别分布于对应的光照单元上，其中，光照单元用于被光源照射时，产生表面等离子体结构光以激发对应的放置单元上的样品中的荧光染料并产生荧光信号。
在本实施例中，周期性分布的样品，例如可以为测序仪中的DNA(脱氧核糖核酸)小球、量子点、荧光纳米小球等。当然本实施例并不具体限定样品的类型，可根据实际需求、实际场景或可能出现的需求和场景进行相应的设定及调整。
在本实施例中，针对周期性阵列排列样品，通过改变入射光照明角度，有效地实现样品选择性被激发，只需至少两幅图即可实现至少2倍分辨率的提升，从而有效地降低了原始图像数量，不需要频域重构算法，较少伪影的产生，不影响样品观测，而且有效地 降低了显微成像装置的搭建难度和制作成本。
具体地，作为一实施例，如图1所示，本实施例提供的显微成像装置，主要包括但并不仅限于光源1、准直透镜2、第一反射镜3、光束角度控制器件4、第一会聚透镜5、第二反射镜6、第二会聚透镜7、用于显微成像装置的光照芯片8、物镜10、筒镜11、成像处理设备12及计算机(图中未示出)。
光源1被配置为向光照芯片8照射光线；准直透镜2用于接收光源1照射的光线，并将准直后的平行光线发射至第一反射镜3；第一反射镜3用于将光线反射至光束角度控制器件4；光束角度控制器件4被配置为从第一反射镜3接收光线，并且发射第一出射角度的出射光至第一会聚透镜5；第二反射镜6用于将经过第一会聚透镜5会聚后的会聚光反射至第二会聚透镜7；第二会聚透镜7用于将第二出射角度(与入射面法线的角度，例如60度)的出射光照射至光照芯片8上，产生表面等离子体结构光，以激发样品9中的荧光染料并产生荧光信号；物镜10用于收集光照芯片8上产生的荧光信号；筒镜11用于将物镜10收集到的荧光信号会聚到成像处理设备12。
成像处理设备12与计算机通信连接，成像处理设备12被配置为根据在不同照射角度下(例如，60度和作为对称角度的-60度)光照芯片8上产生的荧光信号生成至少两张原始图像，并且对至少两张原始图像执行叠加处理以生成超分辨率显微图像。
作为一优选实施方式，光束角度控制器件4被配置为将照射角度的切换时间设定为1ms以下，以使得其切换时间远小于传统结构光显微成像方式中的位移和旋转平台运动所需时间。
作为一可选实施方式，光束角度控制器件4可以使用旋转运动平台带动其反射镜旋转实现光线照射角度的控制，光束角度控制器件4包括扫描振镜，优选可以包括单轴扫描振镜，更具体可以包括XY扫描振镜，但并不具体限定光束角度控制器件4的类型，只要能够实现相应的功能，可根据实际需求、实际场景或可能出现的需求和场景进行相应的设定及调整。
在本实施例中，显微成像装置由于只需要通过扫描振镜改变入射光的照明角度，不需要对光栅进行旋转和平移，因此有效地降低了显微成像装置的搭建难度和制作成本。
在本实施例中，光源1包括激光光源、LED光源及汞灯中的任意一种或多种，但并不具体限定光源1的类型，只要能够实现相应的功能，可根据实际需求、实际场景或可能出现的需求和场景进行相应的设定及调整。
以下具体说明光照芯片8的结构及其上放置的样品9。
如图2及图5所示，光照芯片8(也称纳米芯片)主要包括光照阵列结构(也称纳米 阵列结构)及光照井84(也称纳米井)。
其中，作为一可选实施方式，光照阵列结构主要包括衬底81、基板层(基板层的材料包括二氧化硅)及若干光照单元83，基板层设置于衬底81上，若干光照单元83以正六边形(也可以为正四边形等其他形状)方式周期性设置于基板层内并分布于衬底81上，光照井84设置于基板层上光照单元83延伸的一表面。
作为一优选实施方式，参考图2所示，基板层主要包括第一基板层821及第二基板层822(图2中的虚线仅为了示意而画出，实际并不做物理上的划分，可以通过完全相同的材料构成)。第一基板层821被配置为设置若干光照单元83，第一基板层821的高度与光照单元83的高度相对应，例如80nm；第二基板层822被配置为分布于光照单元83延伸的上表面与光照井84接触基板层的下表面之间，第二基板层822可以起到保护光照单元83的作用，也可有效地起到防腐蚀作用，因此第二基板层822的高度可根据实际需求而设定，例如20nm。
作为一优选实施方式，参考图3及图4所示，光照单元83可以为光照柱体，优选可以为光照圆柱体，光照单元83可以为实心金属，实心金属优选可以为银，也可以为铜、铝、金等。当然本实施例并不具体限定光照单元83的形状及材料，只要能够实现相应的功能，可根据实际需求、实际场景或可能出现的需求和场景进行相应的设定及调整。
参考图2及图6所示，光照井84上划分有用于放置样品9的若干放置单元841，每个放置单元841分别分布于对应的光照单元83上，其中，光照单元83用于被光源照射时，产生表面等离子体结构光以激发对应的放置单元841上的样品9中的荧光染料并产生荧光信号。
作为一优选实施方式，考虑到表面离子效应，参考图6所示，放置单元以对称方式分布于光照单元83的左侧或右侧，其中，光照单元83用于分别被对称照射角度的光源照射时，产生表面等离子体结构光以分别激发分布于光照单元83左右两侧的放置单元841上的样品中的荧光染料。
具体地，本实施例的光照芯片针对周期性分布样品和表面等离子体照明特性做了特殊结构设计。衬底的材料包括透光材料(例如，石英、BK7等高透过率材料)，以便光源透过衬底照射到光照单元上。光照井结构可严格按照光照阵列结构设计，光照井的放置单元的长宽尺寸参数根据光照单元之间的间距确定，光照井的高度根据样品尺寸确定。光照井的材料包括不透光材料，例如TiN(氮化钛)，保证入射光角度变化时产生的等离子体结构光不会激发隔壁样品。
光照单元根据激光波长和分辨率要求，结合有限元分析仿真，选择不同材料和尺寸 参数。例如532nm激光激发下，根据有限元分析仿真，光照单元的材料选择银，其直径为60nm，高度为60nm，Pitch(指相邻两个光照单元(即光照圆柱体)中心之间的距离)为150nm情况下，表面等离子体具有较好的共振能量及符合分辨率提升要求。在不同的光源照射角度下，产生不同的表面等离子体能量分布，参考图7和图8所示，光照单元以白色圈体示出，其内部着色圈体示出激发程度(越靠近内部激发程度越强)，照射角度分别是+60度(对应图7)和-60度(对应图8)，两个对称角度产生的表面等离子体对称分布在光照单元左右两侧，根据这一特性设计上层光照井。
图9示出未照射激光时的光照芯片表面上的样品，图中未激发的样品以黑色圆圈，当激光照射光照芯片时，产生的表面等离子体激发样品中的荧光染料，对称改变照射角度，光照单元左右两侧分布的样品依次被激发，图10示出光照单元左侧样品被激发(结合图6中的光照单元排列方式考虑)，激发的样品以灰色圆圈示出，图11示出光照单元右侧样品被激发(结合图6中的光照单元排列方式考虑)，激发的样品同样以灰色圆圈示出。
以下具体说明利用如上述的显微成像装置获取超分辨率显微图像的使用流程。
在本实施例中，只需要采集两张原始图像，不需要重构算法，直接合成超分辨率显微图像，主要包括以下流程步骤：
A、激发光经准透镜和第一反射镜照射到光束角度控制器件，激光以一定角度出射，如图1所示虚线，该光线经第一会聚透镜会聚后，由第二反射镜照射到第二会聚透镜，最终以一定角度(比如+60度)照射到光照芯片上，激光激发光照阵列结构，产生表面等离子体结构光，如图7所示，等离子体局限在光照单元左侧，光照井结构按照光照阵列结构分布设计，每两个放置单元分布于光照单元左右两侧，如图6所示，因此等离子体结构光只激发光照单元上方左侧放置单元中的样品，被照射样品产生荧光信号，如图10所示(白色表示产生荧光信号)，物镜收集样品产生的荧光信号，经筒镜聚焦在成像处理设备，将光信号转换成电信号后传输到计算机，形成第一张原始图像。
B、控制光束角度控制器件，改变出射光角度，与步骤A中的角度对称，如图1所示实线，激发光最终以一定角度(例如-60度)照射到光照芯片上，激光激发光照阵列结构，产生表面等离子体结构光，如图8所示，等离子体局限在光照单元右侧，激发光照单元上方右侧放置单元中的样品，右侧样品被激发荧光信号，如图11所示，物镜收集样品产生的荧光信号，经筒镜聚焦在成像处理设备，形成第二张原始图像。
C、对采集的两张原始图像进行空间域图像叠加，获得超分辨率显微图像，根据应用不同，进行后续图像处理及相关数据处理。
在本实施例中，涉及到多色染料的情况下，需要触发不同波长激光，即以不同波长激光分别采集原始图像，进行超分辨率显微图像合成，具体采集原始图像及图像合成步骤如上述步骤A至C，每次可重复执行如上述步骤A至C，因此不再一一赘述。
其中，步骤A中最终照射到光照芯片的具体角度需要根据时域有限元分析仿真来确定，跟光照阵列结构的尺寸、材料、入射波长等参数有关，例如，对于532nm激发光源，衬底上铺设60nm高度和60nm直径银圆柱(即光照单元)，光照单元以正六边形周期性分布的情况下，根据时域有限元分析仿真结果建议使用+60度和-60度角度。
光束角度控制器件的出射光角度根据第一会聚透镜和第二会聚透镜构成的透镜组的放大倍率，并结合光照芯片处的角度计算出需要控制的角度，例如，透镜组的放大倍率是2倍，光照芯片照射角度是+60度和-60度，那么光束角度控制器件的出射光角度是±60度/2，即+30度和-30度。
本实施例提供的显微成像装置及其光照芯片，主要具有以下有益效果：
1)只需要控制光束角度控制器件改变出射光角度两次，切换时间通常小于1ms，远小于传统结构光照明显微成像方法中用到的位移和旋转平台运动时间，系统实时性和通量优势明显，并且因此可避免染料光漂白和样品光损伤，特别适用于光敏感染料和生物样品；
2)显微成像图像重建只需要简单的空间域处理，而非复杂的频域算法处理，减少伪影的产生，获得最真实的样品形态结果，这对于定量分析应用非常重要，能有效提高分析结果的准确率，例如二代基因测序中，芯片上DNA小球规则排布，识别的准确性直接影响最终测序结果的可靠性，传统结构光照明显微成像方法中的频域重构算法不可避免的引入伪影，从而影响识别准确性；
3)显微成像装置的光路简单，降低了搭建难度，可兼容常用的显微成像系统，降低了制作成本；
4)输出的显微超分辨率图像理论上可实现至少2倍分辨率的提升。
作为另一实施例，如图12所示，本实施例提供的显微成像装置，主要包括光源1、准直透镜2、反射镜3’、光束角度控制器件4、透镜13、二向色镜14、物镜10、筒镜11、成像处理设备12和计算机(图中未示出)。
其中，光源1、准直透镜2、反射镜3’、光束角度控制器件4、筒镜11、成像处理设备12及计算机的结构及功能可参考如上述实施方式中的相应部件，故不再一一赘述。
在本实施例中，主要将照明光路从如上述的投射式替换为反射式。具体地，参考图12所示，准直透镜2用于接收光源1照射的光线，并将准直后的光线发射至反射镜3’； 反射镜3’用于将光线反射至光束角度控制器件4；光束角度控制器件4被配置为从反射镜3’接收光线，并且发射第一出射角度的出射光至透镜5；透镜5用于将会聚光照射至二向色镜14；二向色镜14用于将接收到的会聚光反射到物镜10；物镜10用于将第三出射角度的出射光照射至光照芯片8上，产生表面等离子体结构光，以激发光照井中的样品9发射荧光信号；物镜10还用于收集光照芯片8上产生的荧光信号，并透过二向色镜14发射到筒镜11；筒镜11用于将接收到的荧光信号会聚到成像处理设备12，成像处理设备12用于获取第一张原始图像；再控制光束角度控制器件4，改变对称的出射光角度，成像处理设备12还用于获取第二张原始图像，叠加两张原始图像，以形成超分辨率显微图像。
本实施例提供的显微成像装置，针对阵列排列生物样品，通过改变入射光照明角度，有效地实现样品选择性被激发，只需至少两幅图即可实现至少2倍分辨率的提升，从而有效地降低了原始图像数量，不需要频域重构算法，较少伪影的产生，不影响样品观测，而且有效地降低了显微成像装置的搭建难度和制作成本。
本公开内容的显微成像装置不局限于二代基因测序，如果应用于二代基因测序系统的话，可以提高测序通量，减少光漂白和光损失，对测序来说也比较重要，而且减少伪影，提高DNB(DNA纳米球)识别率，从而提高Q30(测序数据)数据值。
为了克服目前存在的上述缺陷，作为另一实施例，如图13所示，本实施例提供一种显微成像方法，主要包括以下步骤：
步骤201、生成照射角度调节信号以调节照射角度；
步骤202、通过在不同照射角度下光照芯片上产生的荧光信号来获取至少两张原始图像；
步骤203、对至少两张原始图像执行叠加处理以生成显微图像。
在步骤201中，生成针对如上述的显微成像装置的光源调节机构的照射角度调节信号并输出至光源调节机构，以调节光源向光照芯片照射光线的照射角度。
在步骤202中，获取至少两张原始图像，其中，至少两张原始图像通过在不同照射角度下如上述的用于显微成像装置的光照芯片上产生的荧光信号来生成。
在步骤203中，可利用现有图像叠加处理方式对至少两张原始图像执行叠加处理以生成超分辨率的显微图像，具体不再一一赘述。
本实施例提供的显微成像方法，通过获取至少两张原始图像，即可实现超分辨率显微成像，提高了成像实时性和通量，有效地减少了染料光漂泊和样品光损失；使用空间域重构，非频域重构，减少伪影。
图14为根据本实施例提供的一种电子设备的结构示意图。电子设备包括存储器、处理器及存储在存储器上并可在处理器上执行的计算机程序，处理器执行程序时实现如上实施例中的显微成像方法。图14显示的电子设备30仅仅是一个示例，不应对本公开实施例的功能和使用范围带来任何限制。
如图14所示，电子设备30可以以通用计算设备的形式表现，例如其可以为服务器设备。电子设备30的组件可以包括但不限于：上述至少一个处理器31、上述至少一个存储器32、连接不同系统组件(包括存储器32和处理器31)的总线33。
总线33包括数据总线、地址总线和控制总线。
存储器32可以包括易失性存储器，例如随机存取存储器(RAM)321和/或高速缓存存储器322，还可以进一步包括只读存储器(ROM)323。
存储器32还可以包括具有一组(至少一个)程序模块324的程序/实用工具325，这样的程序模块324包括但不限于：操作系统、一个或者多个应用程序、其它程序模块以及程序数据，这些示例中的每一个或某种组合中可能包括网络环境的实现。
处理器31通过执行存储在存储器32中的计算机程序，从而执行各种功能应用以及数据处理，例如本公开如上实施例中的显微成像方法。
电子设备30也可以与一个或多个外部设备34(例如键盘、指向设备等)通信。这种通信可以通过输入/输出(I/O)接口35进行。并且，模型生成的设备30还可以通过网络适配器36与一个或者多个网络(例如局域网(LAN)，广域网(WAN)和/或公共网络，例如因特网)通信。如图14所示，网络适配器36通过总线33与模型生成的设备30的其它模块通信。应当明白，尽管图中未示出，可以结合模型生成的设备30使用其它硬件和/或软件模块，包括但不限于：微代码、设备驱动器、冗余处理器、外部磁盘驱动阵列、RAID(磁盘阵列)系统、磁带驱动器以及数据备份存储系统等。
应当注意，尽管在上文详细描述中提及了电子设备的若干单元/模块或子单元/模块，但是这种划分仅仅是示例性的并非强制性的。实际上，根据本公开的实施方式，上文描述的两个或更多单元/模块的特征和功能可以在一个单元/模块中具体化。反之，上文描述的一个单元/模块的特征和功能可以进一步划分为由多个单元/模块来具体化。
本实施例还提供了一种计算机可读存储介质，其上存储有计算机程序，程序被处理器执行时实现如上实施例中的显微成像方法中的步骤。
其中，可读存储介质可以采用的更具体可以包括但不限于：便携式盘、硬盘、随机存取存储器、只读存储器、可擦拭可编程只读存储器、光存储器件、磁存储器件或上述的任意合适的组合。
在可能的实施方式中，本公开还可以实现为一种程序产品的形式，其包括程序代码，当程序产品在终端设备上执行时，程序代码用于使终端设备执行实现如上实施例中的显微成像方法中的步骤。
其中，可以以一种或多种程序设计语言的任意组合来编写用于执行本公开的程序代码，程序代码可以完全地在用户设备上执行、部分地在用户设备上执行、作为一个独立的软件包执行、部分在用户设备上部分在远程设备上执行或完全在远程设备上执行。
虽然以上描述了本公开的具体实施方式，但是本领域的技术人员应当理解，这仅是举例说明，本公开的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本公开的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更和修改均落入本公开的保护范围。

Claims (19)

  1. 一种用于显微成像装置的光照芯片，其特征在于，包括：
    光照阵列结构，包括衬底及周期性分布于所述衬底的若干光照单元；以及
    光照井，设置于所述光照阵列结构上所述光照单元延伸的一表面，所述光照井上划分有用于放置样品的若干放置单元，每个所述放置单元分别分布于对应的所述光照单元上，
    其中，所述光照单元用于被光源照射时，产生表面等离子体结构光以激发对应的所述放置单元上的样品中的荧光染料并产生荧光信号。
  2. 如权利要求1所述的光照芯片，所述光照芯片还包括设置于所述衬底上的基板层，所述若干光照单元分别设置于所述基板层内，所述光照井设置于所述基板层上所述光照单元延伸的一表面。
  3. 如权利要求2所述的光照芯片，所述基板层包括：
    第一基板层，被配置为设置所述若干光照单元；以及
    第二基板层，被配置为分布于所述光照单元延伸的上表面与所述光照井接触所述基板层的下表面之间。
  4. 如权利要求2所述的光照芯片，所述基板层的材料包括二氧化硅。
  5. 如权利要求1所述的光照芯片，至少每两个所述放置单元分布于对应的所述光照单元上的两侧。
  6. 如权利要求5所述的光照芯片，所述放置单元以对称方式分布于所述光照单元的左侧或右侧，
    其中，所述光照单元用于分别被对称照射角度的光源照射时，产生所述表面等离子体结构光以分别激发分布于所述光照单元左右两侧的所述放置单元上的样品中的荧光染料。
  7. 如权利要求1所述的光照芯片，若干所述光照单元以正多边形方式周期性分布于所述衬底。
  8. 如权利要求1所述的光照芯片，所述光照单元包括光照柱体。
  9. 如权利要求8所述的光照芯片，所述光照柱体包括光照圆柱体。
  10. 如权利要求1～9中任意一项所述的光照芯片，所述衬底的材料包括透光材料；和/或，
    所述光照单元的材料包括金属材料；和/或，
    所述光照井的材料包括不透光材料。
  11. 一种显微成像装置，其特征在于，包括：
    如权利要求1～10中任意一项所述的用于显微成像装置的光照芯片；
    光源，被配置为向所述光照芯片照射光线；
    光束角度控制器件，被配置为调节所述光源向所述光照芯片照射光线的照射角度；以及
    成像处理设备，被配置为根据在不同照射角度下所述光照芯片上产生的荧光信号生成至少两张原始图像，并且对至少两张原始图像执行叠加处理以生成显微图像。
  12. 如权利要求11所述的显微成像装置，所述显微成像装置还包括准直透镜、反射镜、透镜、二向色镜、物镜及筒镜；
    所述准直透镜用于接收所述光源照射的光线，并将准直后的光线发射至所述反射镜；
    所述反射镜用于将光线反射至所述光束角度控制器件；
    所述光束角度控制器件被配置为从所述反射镜接收所述光线，并且发射第一出射角度的出射光至所述透镜；
    所述透镜用于将会聚光照射至所述二向色镜；
    所述二向色镜用于将接收到的会聚光反射到所述物镜；
    所述物镜用于将第三出射角度的出射光照射至所述光照芯片以产生荧光信号；
    所述物镜还用于收集所述光照芯片上产生的所述荧光信号，并透过所述二向色镜发射到所述筒镜；
    所述筒镜用于将接收到的所述荧光信号会聚到所述成像处理设备。
  13. 如权利要求11所述的显微成像装置，所述显微成像装置还包括准直透镜、第一反射镜、第二反射镜、第一会聚透镜、第二会聚透镜、物镜及筒镜；
    所述准直透镜用于接收所述光源照射的光线，并将准直后的光线发射至所述第一反射镜；
    所述第一反射镜用于将光线反射至所述光束角度控制器件；
    所述光束角度控制器件被配置为从所述第一反射镜接收所述光线，并且发射第一出射角度的出射光至所述第一会聚透镜；
    所述第二反射镜用于将经过所述第一会聚透镜会聚后的会聚光反射至所述第二会聚透镜；
    所述第二会聚透镜用于将第二出射角度的出射光照射至所述光照芯片上；
    所述物镜用于收集所述光照芯片上产生的荧光信号；
    所述筒镜用于将所述物镜收集到的所述荧光信号会聚到所述成像处理设备。
  14. 如权利要求11所述的显微成像装置，所述光束角度控制器件被配置为将照射角度的切换时间设定为1ms以下。
  15. 如权利要求11所述的显微成像装置，所述光束角度控制器件包括扫描振镜。
  16. 如权利要求11～15中任意一项所述的显微成像装置，所述光源包括激光光源、LED光源及汞灯中的任意一种或多种。
  17. 一种显微成像方法，其特征在于，包括：
    获取至少两张原始图像，其中，所述至少两张原始图像通过在不同照射角度下如权利要求1～10中任意一项所述的用于显微成像装置的光照芯片上产生的荧光信号来生成；以及
    对至少两张原始图像执行叠加处理以生成显微图像。
  18. 一种电子设备，包括存储器、处理器及存储在存储器上并可在处理器上执行的计算机程序，其特征在于，所述处理器执行计算机程序时实现如权利要求17所述的显微成像方法。
  19. 一种计算机可读介质，其上存储有计算机指令，其特征在于，所述计算机指令在由处理器执行时实现如权利要求17所述的显微成像方法。
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