CN117368173B - Imaging system and imaging method - Google Patents
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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Abstract
The imaging system comprises a light source assembly, an objective table, a first light beam rotating assembly, an imaging assembly and a second light beam rotating assembly, wherein the objective table is used for bearing a sample to be detected, the first light beam rotating assembly is configured to have a first state and a second state, under the first state, excitation light emitted by the light source assembly irradiates the sample to be detected to excite first linear region fluorescence extending along a first direction, under the second state, excitation light emitted by the light source assembly irradiates the sample to be detected to excite second linear region fluorescence extending along a second direction after passing through the first light beam rotating assembly, the imaging assembly is used for receiving the first linear region fluorescence of a plurality of positions to generate a first area array fluorescent image group of a surface region and receiving the second linear region fluorescence of a plurality of positions to generate a second area array fluorescent image group of the surface region, and super-resolution images are formed according to the first area array fluorescent image group and the second area array fluorescent image group.
Description
Technical Field
The present application relates to the field of gene sequencing and biological sample technology, and more particularly, to an imaging system and imaging method.
Background
The gene sequencer detects four bases of ATCG in the chip in the process of gene sequencing. In general, the higher the data volume density on the chip, the lower the detection cost, and the higher the detection of the high-density chip, the higher the resolution of the gene sequencer, if the density of the DNA clusters on the chip exceeds the resolution of the microscope objective, the higher the resolution of the gene sequencer is needed.
In the related art, an imaging system of the gene sequencer can adopt a structured light illumination super-resolution microscope to realize higher resolution imaging, however, in the traditional structured light illumination scheme, a great amount of energy is wasted in the diffraction process because a diffraction device is required to diffract laser light, meanwhile, fringes generated on a focal plane (sample surface) of a microscope objective are two-dimensionally distributed, when the structured light illumination super-resolution microscope performs line scanning imaging, the ratio of laser energy (single line) actually used for imaging is lower than the ratio of total laser energy (two-dimensional plane) passing through the objective, and enough laser energy is difficult to ensure, and if enough laser energy cannot be ensured, the signal to noise ratio of imaging cannot be ensured, so that the image quality is poor.
Disclosure of Invention
The embodiment of the application provides an imaging system and an imaging method using the imaging system.
An imaging system of an embodiment of the present application for a gene sequencer, comprising:
the light source component is used for emitting excitation light;
the objective table is arranged on the light path of the light source assembly and is used for carrying a sample to be tested;
a first beam rotating assembly configured to have a first state and a second state, the second state being located in an optical path between the light source assembly and the stage; in the first state, the excitation light emitted by the light source assembly irradiates the sample to be detected to excite the fluorescence of a first line area extending along a first direction, and in the second state, the excitation light emitted by the light source assembly irradiates the sample to be detected after passing through the first beam rotating assembly to excite the fluorescence of a second line area extending along a second direction;
the imaging component is used for receiving the fluorescence of the first linear region at a plurality of positions to generate a first area array fluorescence image group of the area region and receiving the fluorescence of the second linear region at a plurality of positions to generate a second area array fluorescence image group of the area region, and reconstructing a two-dimensional super-resolution image according to the first area array fluorescence image group and the second area array fluorescence image group;
A second beam rotating assembly configured to have a first imaging state and a second imaging state, the second imaging state being located in an optical path between the imaging assembly and the stage; in the first imaging state, the imaging assembly receives the first line area fluorescence at a plurality of locations to generate a first area array fluorescence image set of the area, and in the second imaging state, the second beam rotation assembly rotates the second line area fluorescence by 90 degrees and causes the imaging assembly to receive the second line area fluorescence at a plurality of locations to generate a second area array fluorescence image set of the area.
In some embodiments, the first beam rotating assembly is in a first position outside the optical path between the light source assembly and the stage in the first state, and the first beam rotating assembly is in a second position on the optical path between the light source assembly and the stage in the second state.
In some embodiments, a plurality of mirrors are disposed inside the first beam rotating assembly, and a plurality of mirrors and an optical axis of the excitation light form a preset angle.
In some embodiments, the reflecting mirrors include a first reflecting mirror, a second reflecting mirror, a third reflecting mirror, a fourth reflecting mirror, a fifth reflecting mirror and a sixth reflecting mirror, in the second state, the excitation light sequentially passes through the first reflecting mirror, the second reflecting mirror, the third reflecting mirror, the fourth reflecting mirror, the fifth reflecting mirror and the sixth reflecting mirror, the first reflecting mirror is perpendicular to a first plane and forms an included angle of 45 degrees with a second plane, the second reflecting mirror is perpendicular to a third plane and forms an included angle of 22.5 degrees with the first plane, the third reflecting mirror forms an included angle of 45 degrees with the first plane, the second plane and the third plane, the fourth reflecting mirror is perpendicular to the first plane and forms an included angle of 45 degrees with the second plane, the fifth reflecting mirror is perpendicular to the third plane and forms an included angle of 22.5 degrees with the second plane, the fifth reflecting mirror forms an included angle of 45 degrees with the third plane, the second plane and the third plane forms an included angle of 45 degrees with the second plane, and the third plane forms an included angle of 45 degrees with the second plane.
In some embodiments, in the first state, the first mirror and the sixth mirror are out of the optical path of the excitation light.
In some embodiments, the first beam rotating assembly and the second beam rotating assembly are identical in structure.
In some embodiments, the first beam rotating assembly is in a first imaging state and the second beam rotating assembly is in a second imaging state when the first beam rotating assembly is in a first state.
In some embodiments, the stage is coupled to a drive assembly, the drive assembly driving the stage to move in the second direction when in the first state, and the drive assembly driving the stage to move in the first direction when in the second state.
In some embodiments, the imaging system further comprises a control component, when the first beam rotating component is in the first state, the control component controls the driving component to drive the objective table to move along the second direction and controls the light source component to emit the excitation light at a preset switch time sequence, so that the excitation light irradiates the sample to be detected along the second direction to excite fluorescence of a plurality of first line areas, and at least one non-irradiated line area exists among the plurality of irradiated line areas; when the first light beam rotating component is in the second state, the control component controls the driving component to drive the objective table to move along the first direction, controls the light source component to emit the excitation light at a preset switch time sequence, so that the excitation light irradiates the sample to be detected along the second direction to excite a plurality of second line areas to fluoresce, and at least one line area which is not irradiated exists among the plurality of irradiated line areas.
In certain embodiments, the imaging system further comprises a first dichroic mirror for collecting the fluorescence and transmitting the fluorescence to the first dichroic mirror, and an objective for reflecting the excitation light to the sample under test and for transmitting the fluorescence to the imaging assembly.
In certain embodiments, the imaging assembly includes a plurality of second dichroic mirrors that distribute the fluorescence light to a plurality of imaging channels, the second dichroic mirrors being located between the imaging channels and the first dichroic mirrors, each of the imaging channels including a converging lens for converging the fluorescence light to the line camera and a line camera for receiving the fluorescence light.
The embodiment of the application provides an imaging method of an imaging system, the imaging system includes a light source assembly, an objective table, a first light beam rotating assembly and an imaging assembly, a sample to be measured is carried on the objective table, the imaging method includes:
adjusting the first beam rotating component to a first state, and emitting excitation light through the light source component so as to irradiate the sample to be detected and excite fluorescence of a first line area extending along a first direction;
Receiving, by the imaging assembly, the first line area fluorescence to generate a first area array fluorescence image set of one area;
adjusting the first beam rotating component to a second state, and exciting fluorescence of a second line area extending along a second direction by irradiating the sample to be detected with emitted excitation light, wherein the second direction is perpendicular to the first direction;
receiving the second line area fluorescence again through the imaging assembly to generate a second area array fluorescence image set of one area;
and combining the super-resolution image according to the first area array fluorescent image group and the second area array fluorescent image group through the imaging component.
In some embodiments, the method for generating a first area array fluorescent image includes:
the objective table is driven by the driving component to move the sample to be detected along the second direction, meanwhile, the light source component is controlled to enable the excitation light to irradiate the sample to be detected along the second direction and excite a plurality of first line areas to emit fluorescence, at least one first line area which is not irradiated exists among the fluorescence of the first line areas, each first line area is excited to emit fluorescence, and the first area array fluorescence image of one area can be generated by the imaging component.
In some embodiments, the method for generating a second area array fluorescent image includes:
the object stage is driven to move the sample to be detected along the first direction through the driving component, meanwhile, the light source component is controlled to enable the excitation light to irradiate the sample to be detected along the first direction and excite a plurality of second line area fluorescence, at least one non-irradiated second line area exists between the second line area fluorescence, each time the second line area fluorescence is excited, and the second area array fluorescence image of one area can be generated through the imaging component.
In the imaging system of this embodiment, through light source subassembly transmission excitation light to rotate excitation light through the light beam rotary unit and in order to form quadrature excitation light, then gather the image through imaging module respectively, form super-resolution imaging, compare with traditional super-resolution imaging, the energy utilization rate that this scheme utilized the line scanning to realize super-resolution imaging is high, there is not the energy waste, can guarantee sufficient laser energy, be favorable to improving image quality, simultaneously, this scheme utilizes the excitation light of quadrature two directions and forms super-resolution imaging, can promote the resolution in the quadrature direction by a wide margin.
Additional aspects and advantages of embodiments of the application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of embodiments of the application.
Drawings
The foregoing and/or additional aspects and advantages of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a schematic diagram of the main structure of an imaging system of an embodiment of the present application;
FIG. 2 is a block schematic diagram of an imaging system of an embodiment of the present application;
FIG. 3 is a schematic structural view of an imaging system according to an embodiment of the present application;
fig. 4 to 6 are schematic diagrams of an excitation light rotation process according to an embodiment of the present application;
fig. 7 to 8 are schematic views illustrating internal structures of the first beam rotating assembly according to the embodiment of the present application at different angles;
fig. 9 to 11 are two-dimensional imaging schematic diagrams of structural light illumination stripes in the X-direction according to the embodiment of the present application;
fig. 12 to 17 are schematic views showing irradiation of excitation light in a first state by the first beam rotating assembly according to the embodiment of the present application;
fig. 18 to 20 are two-dimensional imaging schematic diagrams of a structured light illumination stripe in the Y direction according to an embodiment of the present application;
Fig. 21 to 26 are schematic views showing irradiation of excitation light by the first beam rotating assembly in the second state according to the embodiment of the present application;
fig. 27 is a flow chart of an imaging method of an embodiment of the present application.
Description of main reference numerals: imaging system 100, light source assembly 10, excitation light 11, laser emitter 12, beam shaper 13, multimode optical fiber 14, stage 20, first beam rotating assembly 30, first mirror 31, second mirror 32, third mirror 33, fourth mirror 34, fifth mirror 35, sixth mirror 36, beam inlet 37, beam outlet 38, imaging assembly 40, second dichroic mirror 41, imaging channel 42, converging lens 421, linear camera 422, optical filter 423, sample 50 to be measured, first line area fluorescence 51, second line area fluorescence 52, imaging area 53, first line area 54, second line area 55, first dichroic mirror 61, objective lens 62, driving assembly 70, control assembly 80, second beam rotating assembly 90.
Detailed Description
Embodiments of the present application are further described below with reference to the accompanying drawings. The same or similar reference numbers in the drawings refer to the same or similar elements or elements having the same or similar functions throughout.
In addition, the embodiments of the present application described below in conjunction with the drawings are exemplary only and are not to be construed as limiting the present application.
In this application, unless expressly stated or limited otherwise, a first feature "up" or "down" a second feature may be the first and second features in direct contact, or the first and second features in indirect contact via an intervening medium. Moreover, a first feature being "above," "over" and "on" a second feature may be a first feature being directly above or obliquely above the second feature, or simply indicating that the first feature is level higher than the second feature. The first feature being "under", "below" and "beneath" the second feature may be the first feature being directly under or obliquely below the second feature, or simply indicating that the first feature is less level than the second feature.
In the sequencing process of the gene sequencer, fluorescent imaging is needed to be carried out on four bases of ATCG respectively, and as each target point to be detected is in a submicron scale range, the gene sequencer can inevitably adopt a microscopic imaging mode to realize the detection purpose. An important index of a gene sequencer is the dot density on the chip, which determines the amount of data on a chip, and also determines the cost of production of unit data. Since the difference between the dot density and the amount of the reagent used per unit area is not large, the higher the dot density is, the lower the detection cost is.
High density chips require coordination of higher resolution microscopy systems. However, conventional wide field microscope systems have diffraction limits and resolution is typically up to around 200 nm. In order to achieve higher resolution and thus higher chip dot density, it is necessary to implement this by means of "super-resolution" optics.
In the field of gene sequencing, imaging speed is an important indicator that determines the time to output a sequencing report. In order to further increase the imaging detection speed, a scheme of adopting line scanning imaging is a trend in the future. In the conventional SIM scheme, diffraction and light splitting are required by devices such as a grating, a spatial light modulator, a digital micro-mirror (DMD) and the like, and diffracted ±1st-order light beams interfere on the focal plane (sample plane) of an objective lens to generate sine fringes to form structured light illumination. Or the later-occurring non-diffraction spectroscopic scheme realized by optical devices such as a spectroscope, a prism and the like can also form interference fringes on the focal plane (sample surface) of the objective lens.
However, the greatest problem of the conventional scheme is that its energy utilization rate is extremely low. Firstly, the diffraction beam splitting scheme is that the energy utilization rate is limited by the diffraction efficiency of a diffraction device, and the energy cannot be completely transmitted to the back focal plane of the objective lens, so that the energy is greatly wasted. Second, the fringes produced at the focal plane of the objective lens (sample plane) are two-dimensionally distributed, either in a diffractive spectroscopic or non-diffractive spectroscopic scheme. For line scan imaging systems, the ratio of the laser energy actually used for imaging (single line) is also very low compared to the total laser energy (two-dimensional plane) through the objective lens. The imaging speed of the line scanning imaging system is far faster than that of the traditional surface scanning imaging system, and the result is that the exposure time of the single pixel of the line camera is necessarily far less than that of the single pixel of the surface scanning imaging system. If sufficient laser energy is not ensured, the signal to noise ratio of the imaging is not ensured, resulting in poor image quality.
In view of this, referring to fig. 1 to 27, an imaging system 100 is provided in an embodiment of the present application, the imaging system 100 is used for a gene sequencer, and the imaging system 100 includes a light source assembly 10, a stage 20, a first beam rotating assembly 30, an imaging assembly 40, and a second beam rotating assembly 90.
The light source assembly 10 is configured to emit excitation light 11, the objective table 20 is disposed on an optical path of the light source assembly 10, the objective table 20 carries a sample 50 to be measured, the first beam rotating assembly 30 is configured to have a first state and a second state, the second state is located on an optical path between the light source assembly 10 and the objective table 20, in the first state, the excitation light 11 emitted by the light source assembly 10 irradiates the sample 50 to be measured to excite a first linear region fluorescence 51 extending along a first direction, in the second state, the excitation light 11 emitted by the light source assembly 10 irradiates the sample 50 to be measured after passing through the first beam rotating assembly 30 to excite a second linear region fluorescence 52 extending along a second direction, the imaging assembly 40 is configured to receive the first linear region fluorescence 51 at a plurality of positions to generate a first linear fluorescent image group of the surface region, and to receive the second linear region fluorescence 52 at a plurality of positions to generate a second linear fluorescent image group of the surface region, and reconstruct a two-dimensional super-resolution image according to the first linear fluorescent image group and the second linear region fluorescence image group, and the second beam rotating assembly 90 is configured to have a second linear region fluorescence image group of the second linear region fluorescence group extending along the first direction and the second linear region fluorescence image group; in a first imaging state, imaging assembly 40 receives first line area fluorescence 51 at a plurality of locations to generate a first area fluorescence image of the area, and in a second imaging state, second beam rotation assembly 90 rotates second line area fluorescence 52 by 90 degrees and causes imaging assembly 40 to receive second line area fluorescence 52 at a plurality of locations to generate a second area fluorescence image of the area.
In the imaging system 100 of this embodiment, the excitation light 11 is emitted through the light source assembly 10, and the excitation light 11 is rotated through the light beam rotation unit to form the orthogonal excitation light 11, then the images are collected through the imaging assembly 40 respectively to form super-resolution imaging, compared with traditional super-resolution imaging, the energy utilization rate of the super-resolution imaging is high by utilizing line scanning, no energy is wasted, enough laser energy can be ensured, and the image signal to noise ratio is improved.
Specifically, the sample 50 to be measured is a biological sample with fluorescent dye, the sample is arranged on a gene sequencing chip, the gene sequencing chip is placed on the objective table 20, when the first beam rotating assembly 30 is in a first state, the excitation light 11 emitted by the light source assembly 10 irradiates the sample 50 to be measured to form a first light spot, the light spot excites the fluorescent dye on the biological sample to form first line area fluorescence 51, both the light spot and the first line area fluorescence 51 extend towards a first direction X, when the first beam rotating assembly 30 is in a second state, the excitation light 11 emitted by the light source assembly 10 irradiates the sample 50 to be measured to form a second light spot, and when the second light spot excites the fluorescent dye on the biological sample to form second line area fluorescence 52, both the second light spot and the second area fluorescence extend towards a second direction Y.
Referring to fig. 3, in some embodiments, the imaging system 100 further comprises a first dichroic mirror 61 and an objective lens 62, the objective lens 62 for collecting fluorescence and transmitting the fluorescence to the first dichroic mirror 61, the first dichroic mirror 61 for reflecting excitation light 11 to the sample 50 to be measured and for transmitting the fluorescence to the imaging assembly 40.
In some embodiments, the light source assembly 10 includes a laser emitter 12 and a beam shaper 13, the laser emitter 12 is used for emitting laser light, the beam shaper 13 is used for shaping the laser light into excitation light 11, and in particular, the laser emitter 12 can emit laser light with a specific wavelength to excite the sample 50 to be measured to generate fluorescence; the light source assembly 10 may further include a multimode optical fiber 14, the multimode optical fiber 14 being positioned between the laser transmitter 12 and the beam shaper 13, the laser transmitter 12 being connected to the beam shaper 13 by the multimode optical fiber 14.
In certain embodiments, imaging assembly 40 comprises a plurality of second dichroic mirrors 41, the plurality of second dichroic mirrors 41 distributing fluorescence light to a plurality of imaging channels 42, the second dichroic mirrors 41 being located between the imaging channels 42 and the first dichroic mirrors 61, each imaging channel 42 comprising a converging lens 421 and a line camera 422, the converging lens 421 for converging fluorescence light to the line camera 422, the line camera 422 for receiving fluorescence light.
Specifically, since the photosensitive element of the line camera 422 is one-dimensional, only one dimension of the sample can be imaged when the line camera 422 is not scanned, when the first beam rotating component 30 rotates the excitation light 11 by 90 ° to form the second line area fluorescence 52 extending along the second direction Y, the line camera 422 needs to be rotated at this time, otherwise, the second line area fluorescence 52 cannot be identified and imaged at this time, so that the second beam rotating component 90 is disposed on the optical path between the imaging component 40 and the stage 20, the second beam rotating component 90 rotates the second line area fluorescence 52 and transmits the second line area fluorescence 52 to the line camera 422, and the extending direction of the second line area fluorescence 52 after rotation is the first direction X, so that the line camera 422 can perform detection and identification at this time without rotating the line camera 422, thereby simplifying the operation difficulty.
In some embodiments, the imaging assembly 40 includes a filter 423, the filter 423 being positioned between the converging lens 421 and the line camera 422, the filter 423 being configured to filter fluorescence.
Referring to fig. 3, in some embodiments, in the first state, the first beam rotating element 30 is located at a first position, the first position is located outside the optical path between the light source element 10 and the stage 20, in the second state, the first beam rotating element 30 is located at a second position, the second position is located on the optical path between the light source element 10 and the stage 20, specifically, the first beam rotating element 30 is movably disposed at the first position and the second position, when the first beam rotating element 30 is located at the first position, the excitation light 11 does not enter the first beam rotating element 30 and directly irradiates the stage 20, when the first beam rotating element 30 is located at the second position, the excitation light 11 enters the first beam rotating element 30, and when the first beam rotating element 30 rotates the excitation light 11 by 90 ° and irradiates the stage 20.
In some embodiments, the first beam rotating assembly 30 is internally provided with a plurality of mirrors, and the plurality of mirrors and the optical axis of the excitation light 11 form a preset angle, specifically, the excitation light 11 enters from the beam inlet 37 of the first beam rotating assembly 30, rotates the excitation light 11 by reflection of the plurality of mirrors, and then exits from the beam outlet 38.
Referring to fig. 7 and 8, in some embodiments, the mirrors include a first mirror 31, a second mirror 32, a third mirror 33, a fourth mirror 34, a fifth mirror 35, and a sixth mirror 36, in the second state, the excitation light 11 sequentially passes through the first mirror 31, the second mirror 32, the third mirror 33, the fourth mirror 34, the fifth mirror 35, and the sixth mirror 36, the first mirror 31 is perpendicular to the first plane and forms an angle of 45 degrees with the second plane, the second mirror 32 is perpendicular to the third plane and forms an angle of 22.5 degrees with the first plane, the third mirror 33 forms an angle of 45 degrees with the first plane, the second plane, and the third plane, the fourth mirror 34 is perpendicular to the first plane and forms an angle of 45 degrees with the second plane, the fifth mirror 35 is perpendicular to the third plane and forms an angle of 22.5 degrees with the second plane, and the sixth mirror 32 is perpendicular to the third plane, and forms an angle of 45 degrees with the first plane, the second plane, the third plane, and the third plane are perpendicular to the first plane, the second plane, and the third plane are perpendicular to each other.
Referring to fig. 4 to 6 and fig. 7 and 8, specifically, the direction in which the optical axis of the excitation light 11 is located is taken as the third direction Z, the first plane is XOY, the second plane is ZOY, and the third plane is XOZ, as shown in fig. 4, when the light shaped by the beam shaper 13 enters the beam rotation optical path system through the beam inlet 37, the light intensity distribution of the light is an ellipse with the major axis along the X axis direction, the excitation light 11 enters the first excitation light 11 rotation component from the beam inlet 37, and is reflected by the first reflector 31, and the included angle between the normal line of the plane in which the first reflector 31 is located and the optical axis of the incident excitation light 11 on the XOZ plane is 45 °; the excitation light 11 reaches the second reflecting mirror 32 after being reflected, and the included angle between the normal line of the plane where the second reflecting mirror 32 is positioned and the optical axis of the incident excitation light 11 on the XOY plane is 22.5 degrees; the excitation light 11 is reflected by the second reflector 32 to the third reflector 33, wherein the normal line of the plane of the third reflector 33 forms an included angle of 45 degrees with the optical axis of the incident excitation light 11 on the XOZ plane; as shown in fig. 5, the light spot intensity distribution after reflection of the excitation light 11 is still elliptical, but the major axis direction of the ellipse is changed, and an included angle of 45 ° is formed between the major axis direction and the X axis; the excitation light 11 is reflected by the fourth reflecting mirror 34, wherein an included angle between the normal line of the plane of the fourth reflecting mirror 34 and the optical axis of the incident excitation light 11 on the XOZ plane is 45 degrees, the excitation light 11 reaches the fifth reflecting mirror 35, and an included angle between the normal line of the plane of the fifth reflecting mirror 35 and the optical axis of the incident excitation light 11 on the XOY plane is 22.5 degrees; the fifth mirror 35 then reflects the excitation light 11 onto the sixth mirror 36, the normal to the plane of the sixth mirror 36 and the optical axis of the incident excitation light 11 form an angle of 45 ° in the XOZ plane, and finally the sixth mirror 36 reflects the excitation light 11 out of the first beam rotating assembly 30 along the beam outlet 38. The spot intensity after reflection by the sixth mirror 36 is still elliptical as shown in fig. 6, the major axis of the ellipse being parallel to the Y-axis, i.e. the spot intensity distribution of the excitation light 11 at the beam outlet 38 is rotated 90 ° around the Z-axis direction compared to the intensity distribution of the excitation light 11 at the beam inlet 37. Moreover, by reasonably designing the spatial positions of the reflecting mirrors, the excitation light 11 at the beam outlet 38 can be ensured to exit along the front of the excitation light 11 at the beam inlet 37 in the Z-axis direction, that is, the moving-in and moving-out of the rotary optical path system of the excitation light 11 can be ensured not to influence the original directivity of the excitation light 11.
Referring to fig. 7 to 8, in some embodiments, in the first state, the first mirror 31 and the sixth mirror 36 are separated from the optical path of the excitation light 11, specifically, the first beam rotating assembly 30 is fixedly disposed on the optical path between the light source assembly 10 and the stage 20, the first beam rotating assembly 30 is in the first state, the first mirror 31 and the sixth mirror 36 are moved out of the optical path, and the excitation light 11 enters the first beam rotating assembly 30 from the beam inlet 37 and is not reflected directly from the beam outlet 38; in the second state of the first beam rotating assembly 30, the first mirror 31 and the sixth mirror 36 move into the optical path, and the excitation light 11 enters the first beam rotating assembly 30 from the beam entrance 37 and needs to be reflected and then exits from the beam exit 38. Alternatively, the first mirror 31 and the sixth mirror 36 may be moved in a rotational, overturning or translational manner, so that it is not necessary to set two positions for the first beam rotating assembly 30, which is advantageous for reducing the volume of the imaging system 100.
In certain embodiments, the length of the imaging of the first and second line area fluorescence 51, 52 on the photosensitive elements of the imaging assembly 40 is less than or equal to the length of the photosensitive elements, and the width of the imaging of the first and second line area fluorescence 51, 52 on the photosensitive elements of the imaging assembly 40 is less than or equal to the width of the pixels of the photosensitive elements of the imaging assembly 40. Specifically, for example, in the case where the first beam rotating component 30 is in the first state, the first line area fluorescence 51 has a width larger than the width of the pixels of the photosensitive element, the first line area fluorescence 51 may interfere with the imaging effect of the adjacent area, so as to reduce the frequency of the stripes of the first area array fluorescence image, and reduce the resolution of the synthesized super-resolution image. Therefore, the width of the image formed by the first line area fluorescence 51 and the second line area fluorescence 52 on the photosensitive element of the image forming unit 40 is smaller than or equal to the width of the pixel of the photosensitive element of the image forming unit 40, and the image forming effect is optimal.
Referring to fig. 3 and fig. 7 to fig. 8, in some embodiments, the internal structure of the second beam rotating element 90 is the same as that of the first beam rotating element 30, specifically, six mirrors are disposed in the second beam rotating element 90, and the mounting positions of the six mirrors are the same as those of the first beam rotating element 30, and in other embodiments, the second beam rotating element 90 may be other structures that can achieve the same effect.
In some embodiments, when the first beam rotating element 30 is in the first state, the second beam rotating element 90 is in the first imaging state, and when the first beam rotating element 30 is in the second state, the second beam rotating element 90 is in the second imaging state, and in particular, the first beam rotating element 30 is linked with the second beam rotating element 90, and the first beam rotating element 30 and the second beam rotating element can be driven by the same driving mode, which is beneficial to reducing the operation difficulty.
Referring to fig. 2, in some embodiments, the stage 20 is connected to a driving component 70, and the driving component 70 drives the stage 20 to move along the second direction when the first beam rotating component 30 is in the first state, and the driving component 70 drives the stage 20 to move along the first direction when the first beam rotating component 30 is in the second state.
Referring to fig. 2 and 3, in some embodiments, the imaging system 100 further includes a control component 80, when the first beam rotating component 30 is in the first state, the control component 80 controls the driving component 70 to drive the stage 20 to move along the second direction and controls the light source component 10 to emit the excitation light 11 at a preset switching sequence, so that the excitation light 11 irradiates the sample 50 to be measured along the second direction to excite the fluorescent light 51 of the first line areas, and at least one non-irradiated line area exists between the irradiated line areas; when the first beam rotating assembly 30 is in the second state, the control assembly 80 controls the driving assembly 70 to drive the stage 20 to move along the first direction, and controls the light source assembly 10 to emit the excitation light 11 at a preset switching sequence, so that the excitation light 11 irradiates the sample 50 to be measured along the second direction to excite the fluorescence 52 of the plurality of second line areas, and at least one non-irradiated line area exists between the plurality of irradiated line areas.
Referring to fig. 2 and 3, in particular, the control unit 80 may control the movement of the stage 20, and thus the movement of the sample 50 to be measured, by the driving unit 70. While keeping the light source assembly 10 stationary, the control assembly 80 controls the stage 20 to move in the second direction Y, i.e., the sample 50 to be measured moves in the second direction Y relative to the excitation light 11, so that the plurality of line areas sweep the irradiation area in the second direction Y. It will be appreciated that the excitation light 11 may illuminate the sample 50 to be measured in a direction perpendicular to the surface of the sample 50 to be measured, or may illuminate the sample 50 to be measured in other directions, while maintaining the spot of the excitation light 11 on the sample 50 to be measured extending in the first direction X.
In the case that the preset switching timing is in the on period, the light source assembly 10 emits excitation light 11 to cause the sample 50 to be measured to excite the first line area fluorescence 51 or the second line area fluorescence 52; in the case where the preset switching timing is in the off period, the light source assembly 10 turns off the excitation light 11 to form the first line region 54 or the second line region 55 that is not irradiated.
Referring to FIG. 3, in some embodiments, the number of imaging channels 42 is 4, and in particular, the gene sequencer needs to perform fluorescence imaging on four ATCG bases during the sequencing process, and the 4 imaging channels 42 correspond to the four ATCG bases respectively.
In some embodiments, the number of the second dichroic mirrors 41 is three, specifically, the three second dichroic mirrors 41 are different types of dichroic mirrors, further, the coating films of the three second dichroic mirrors 41 are different, four types of ATCG bases form four types of fluorescence, and at least three second dichroic mirrors 41 are required for separating the four types of fluorescence one by one.
Referring to fig. 3, specifically, the optical path of the excitation light 11 is as follows: the laser light emitted from the laser emitter 12 is transmitted into the beam shaper 13 through the multimode optical fiber 14 to form excitation light 11, and the excitation light 11 is reflected by the first dichroic mirror 61 and irradiated onto the sample 50 to be measured through the objective lens 62. Fluorescence excited by the excitation light 11 is collected by the objective lens 62, transmitted through the first dichroic mirror 61, and then is divided into four paths by the three second dichroic mirrors 41 to be transmitted to the four line cameras 422, and the four bases of the ATCG are imaged respectively; then, the first beam rotating unit 30 is adjusted to rotate the excitation light 11, and the excitation light 11 is reflected by the first dichroic mirror 61 and irradiated onto the sample 50 to be measured through the objective lens 62. The fluorescence excited by the excitation light 11 is collected by the objective lens 62, transmitted through the first dichroic mirror 61, and then split into four paths by the three second dichroic mirrors 41, and transmitted to the four line cameras 422, where the four ATCG bases are imaged again, respectively.
Referring to fig. 2 and 27, an imaging method of an imaging system 100 is provided in an embodiment of the present application, where the imaging system 100 includes a light source assembly 10, a stage 20, a first beam rotating assembly 30, and an imaging assembly 40, and the stage 20 carries a sample 50 to be measured, and the imaging method includes:
step 01: the first beam rotating assembly 30 is adjusted to a first state, and excitation light 11 is emitted by the light source assembly 10 to irradiate the sample 50 to be measured to excite first line area fluorescence 51 extending along a first direction;
step 02: receiving, by imaging assembly 40, first line area fluorescence 51 to generate a plurality of first area array fluorescence images of a surface area;
step 03: adjusting the first beam rotating member 30 to a second state, and exciting a second line area fluorescence 52 extending in a second direction by irradiating the sample 50 to be measured with the emitted excitation light 11; the second direction is perpendicular to the first direction;
step 04: receiving second line area fluorescence 52 again through imaging assembly 40 to generate a plurality of second area array fluorescence images of an area;
step 05: the super-resolution image is synthesized from the plurality of first area array fluorescent images and the plurality of second area array fluorescent images by the imaging assembly 40.
Specifically, the imaging system 100 further includes a control component 80, where the control component 80 adjusts the first beam rotating component 30 to a first state, and emits excitation light 11 through the light source component 10 to irradiate the sample 50 to be measured to excite first line area fluorescence 51 extending along the first direction, and then the control component 80 controls the imaging component 40 to receive the first line area fluorescence 51 to generate a plurality of first area array fluorescence images of one area; the control assembly 80 then adjusts the first beam rotating assembly 30 to a second state and irradiates the sample 50 to be measured by emitting excitation light 11 to excite the second line area fluorescence 52 extending in the second direction; the second direction is perpendicular to the first direction; the control assembly 80 again receives the second line area fluorescence 52 through the imaging assembly 40 to generate a plurality of second area array fluorescence images of a surface area; finally, the super-resolution image is synthesized by the imaging component 40 according to the plurality of first area array fluorescent images and the plurality of second area array fluorescent images.
Further, the method for generating the first area array fluorescent image comprises the following steps:
the stage 20 is driven by the driving component 70 to move the sample 50 to be tested along the second direction, meanwhile, the light source component 10 is controlled to enable the excitation light 11 to irradiate the sample 50 to be tested along the second direction and excite a plurality of first line area fluorescence lights 51, at least one first line area 54 which is not irradiated exists among the plurality of first line area fluorescence lights 51, and each first line area fluorescence light 51 is excited, a first area array fluorescence image of one area can be generated by the imaging component 40.
The method for generating the second area array fluorescent image comprises the following steps:
the stage 20 is driven by the driving component 70 to move the sample 50 to be tested along the first direction, meanwhile, the light source component 10 is controlled to enable the excitation light 11 to irradiate the sample 50 to be tested along the first direction and excite a plurality of second line area fluorescence 52, at least one non-irradiated second line area 55 exists among the plurality of second line area fluorescence 52, and each time one second line area fluorescence 52 is excited, a second area array fluorescence image of one area can be generated by the imaging component 40.
Referring to fig. 9-11, in some embodiments, imaging is accomplished by illuminating the sample with periodic stripes of alternating light and dark. In order to achieve the imaging effect, it is also necessary to perform three-step phase shift on the fringes in a dimension that improves the imaging resolution, and then reconstruct the captured images of three different illumination fringe phases, thereby obtaining a super-resolution image.
Specifically, as shown in fig. 9 to 11, a two-dimensional imaging schematic diagram of a structural light illumination stripe in the X direction is shown, which includes a structural light stripe bright area, i.e., an illuminated portion, and a structural light stripe dark area, i.e., a non-illuminated portion. First, the first image is completed by illuminating the A, B, C and G, H, I columns with the structured-light illumination stripes. Thereafter, the structured light illumination stripe changes phase, and irradiates on the C, D, E and I, J, K columns, completing the second image. Finally, the structured-light illumination stripe is again phase-shifted, illuminating on E, F, G and K, L, A columns, completing the third image. And reconstructing the three images to obtain resolution improvement of the dimension perpendicular to the image in the stripe direction.
Referring to fig. 9 to 11, in order to generate the fringe imaging effect in the figures, we need to control the light source assembly 10 at a preset switching timing, specifically, control the laser emitter 12 at the preset switching timing through the control assembly 80. As shown in fig. 12 to 17, the first beam rotating element 30 is schematically illustrated as a stripe generation mode when the excitation light 11 irradiates the sample 50 to be measured in the first state, wherein the first line area fluorescence 51 is an illumination area, and the first line area 54 is an non-illumination area.
Since the detector of the line camera 422 is one-dimensional, only one dimension of the sample can be imaged when not scanning. As shown in fig. 12 to 17, the line camera 422 can perform imaging only in the X direction, and then scan to complete two-dimensional imaging in cooperation with movement of the motorized stage 20 in the Y direction.
Referring to fig. 9 to 11, in order to achieve the same illumination effect as the illumination fringe period in the illumination mode of the excitation light 11, the switching timing of the laser is preset.
Referring to fig. 12 to 17, specifically, the control component 80 controls the laser emitter 12 to emit the excitation light 11 to irradiate column a so that column a excites the first line area fluorescence 51, at this time, the imaging area 53 of the line camera 422 is also located in column a and generates the first line area fluorescence image, after imaging, the control component 80 controls the driving component 70 to drive the stage 20 to move along the Y direction, and repeat the above operations in columns B and C, when the stage 20 moves to column D, the control component 80 controls the laser emitter 12 to emit and close, and continues to move the stage 20, while the line camera 422 images in column D, E, F, so as to complete the imaging of the first line area fluorescence image in a complete fringe period, and repeating the above steps until the imaging of the first line area fluorescence image of the whole sample to be measured is completed.
Referring to fig. 18 to 20, after the above-mentioned imaging is completed, the structural light illumination stripe needs to be rotated by 90 ° to form a structural light illumination stripe in the Y direction, and imaging is performed again, so as to finally realize the orthogonal line scanning super-resolution imaging, and the structural light illumination stripe in the Y direction also needs to be subjected to three-step phase shift.
Specifically, after all the first area fluorescent images of the first beam rotating element 30 in the first state are imaged, the first beam rotating element 30 is adjusted to the second state, and at this time, the stripe formation mode when the excitation light 11 irradiates the sample 50 to be measured is as shown in fig. 18 to 20, wherein the gray area is an illumination area, i.e. the second line area fluorescent light 52, and the white area is an non-illumination area, i.e. the second line area 55.
Since the first beam rotating assembly 30 is in the second state and the second beam rotating assembly 90 is also in the second state, the second beam rotating assembly 90 rotates the second fluorescence light 52 in the second linear region and then images the second fluorescence light by the linear camera 422, so that the linear camera 422 does not need to be rotated, and the linear camera 422 performs imaging in the Y direction, and the two-dimensional imaging is completed by matching with the movement of the electric stage 20 in the X direction.
Referring to fig. 18 to 20, in order to achieve the same illumination effect as one illumination stripe period in the drawings in the illumination mode of the excitation light 11, the switching timing needs to be preset by laser.
Referring to fig. 21 to 26, specifically, the control component 80 controls the laser emitter 12 to emit the excitation light 11 to irradiate the a-column to excite the first linear region fluorescence 51 in the a-column, and the imaging region 53 of the linear camera 422 is located in the a-row to generate the second linear fluorescent image, and the control component 80 controls the driving component 70 to drive the stage 20 to move along the X-direction and repeat the above operations in the B-row and the C-row after imaging, when the stage 20 moves to the D-row, the control component 80 controls the laser emitter 12 to emit to close, and continues to move the stage 20, and simultaneously the linear camera 422 images in the D, E, F-row, so as to complete the imaging of the second linear fluorescent image in a complete stripe period, and repeat the above steps until the imaging of the second linear fluorescent image of the whole sample to be measured is completed.
So far, a first area array fluorescent image and a second area array fluorescent image of the whole sample to be detected are obtained, and all the first area array fluorescent image and the second area array fluorescent image are synthesized to obtain final super-resolution imaging.
Specifically, referring to fig. 9 to 11 and 12 to 17, or referring to fig. 18 to 20 and 21 to 26, in the preset switching timing, one on period and one off period constitute one minimum period of the preset switching timing. The duration T of one on period and one off period is the same and may correspond to the time required for the irradiation region to sweep through three line regions.
For example, the duration T of one on period or one off period may satisfy:
T=3*S/M/V，
where S is the pixel size of the imaging assembly 40, M is the magnification of the imaging system 100, and V is the movement speed of the sample 50 to be measured.
In some embodiments of the present invention, the module involved may be a single-chip microcomputer chip, integrated with a processor, a memory, a communication module, etc. The processor may be a central processing unit (Central Processing Unit, CPU), but may also be other general purpose processors, digital signal processors (Digital Signal Processor, DSP), application specific integrated circuits (Application Specific Integrated Circuit, ASIC), off-the-shelf programmable gate arrays (Field Programmable Gate Array, FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, or the like.
Any process or method descriptions in flow charts or otherwise described herein may be understood as representing modules, segments, or portions of code which include one or more executable instructions for implementing specific logical functions or steps of the process, and further implementations are included within the scope of the preferred embodiment of the present invention in which functions may be executed out of order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, as would be understood by those reasonably skilled in the art of the present invention.
Logic and/or steps represented in the flowcharts or otherwise described herein, e.g., a ordered listing of executable instructions for implementing logical functions, can be embodied in any computer-readable medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, system that includes a processing module, or other system that can fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions.
In the description of the present specification, reference to the terms "certain embodiments," "one embodiment," "some embodiments," "an exemplary embodiment," "an example," "a particular example," or "some examples" means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" is at least two, for example two, three, unless explicitly defined otherwise.
Although embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application, which is defined by the claims and their equivalents.
Claims (11)
1. An imaging system for a genetic sequencer, the imaging system comprising:
the light source component is used for emitting excitation light;
the objective table is arranged on the light path of the light source assembly and is used for carrying a sample to be tested;
a first beam rotating assembly configured to have a first state and a second state, the second state being located in an optical path between the light source assembly and the stage; in the first state, the first beam rotating assembly is located at a first position, the first position is located outside a light path between the light source assembly and the objective table, excitation light emitted by the light source assembly irradiates the sample to be tested to excite fluorescence in a first linear region extending along a first direction, in the second state, the first beam rotating assembly is located at a second position, the second position is located on the light path between the light source assembly and the objective table, the excitation light emitted by the light source assembly irradiates the sample to be tested to excite fluorescence in a second linear region extending along a second direction through the first beam rotating assembly, the first beam rotating assembly is provided with a plurality of reflecting mirrors, the plurality of reflecting mirrors and an optical axis of the excitation light form a preset angle, the reflecting mirrors comprise a first reflecting mirror, a second reflecting mirror, a third reflecting mirror, a fifth reflecting mirror and a sixth reflecting mirror, in the second state, the excitation light sequentially passes through the first reflecting mirror, the second reflecting mirror, the third reflecting mirror, the fifth reflecting mirror and the fifth reflecting mirror form an included angle with the fifth reflecting mirror, the fifth reflecting mirror is perpendicular to the first plane 45, the third reflecting mirror is perpendicular to the fifth reflecting mirror, the fifth reflecting mirror forms a plane 45, the plane 45 is perpendicular to the first plane 45, the plane is formed by the first plane 45, the included angles formed by the first plane, the second plane and the third plane of the sixth reflecting mirror are 45 degrees, and the first plane, the second plane and the third plane are perpendicular to each other;
The imaging component is used for receiving the fluorescence of the first linear region at a plurality of positions to generate a first area array fluorescence image group of the area region and receiving the fluorescence of the second linear region at a plurality of positions to generate a second area array fluorescence image group of the area region, and reconstructing a two-dimensional super-resolution image according to the first area array fluorescence image group and the second area array fluorescence image group;
a second beam rotating assembly configured to have a first imaging state and a second imaging state, the second imaging state being located in an optical path between the imaging assembly and the stage; in the first imaging state, the imaging assembly receives the first line area fluorescence at a plurality of locations to generate a first area array fluorescence image set of the area, and in the second imaging state, the second beam rotation assembly rotates the second line area fluorescence by 90 degrees and causes the imaging assembly to receive the second line area fluorescence at a plurality of locations to generate a second area array fluorescence image set of the area.
2. The imaging system of claim 1, wherein in the first state, the first mirror and the sixth mirror are out of the optical path of the excitation light.
3. The imaging system of claim 1, wherein the first beam rotating assembly and the second beam rotating assembly are identical in structure.
4. The imaging system of claim 1, wherein the first beam rotating assembly is in a first imaging state and the second beam rotating assembly is in a second imaging state when the first beam rotating assembly is in a second state.
5. The imaging system of claim 1, wherein the stage has a drive assembly coupled thereto, the drive assembly driving the stage to move in the second direction when in the first state, and the drive assembly driving the stage to move in the first direction when in the second state.
6. The imaging system of claim 5, further comprising a control assembly that controls the drive assembly to drive the stage to move in the second direction and controls the light source assembly to emit the excitation light at a preset switching timing when the first beam rotation assembly is in the first state such that the excitation light irradiates the sample to be measured in the second direction to excite fluorescence of a plurality of the first line regions, at least one of the line regions not irradiated being present between the plurality of the irradiated line regions; when the first light beam rotating component is in the second state, the control component controls the driving component to drive the objective table to move along the first direction, controls the light source component to emit the excitation light at a preset switch time sequence, so that the excitation light irradiates the sample to be detected along the second direction to excite a plurality of second line areas to fluoresce, and at least one line area which is not irradiated exists among the plurality of irradiated line areas.
7. The imaging system of claim 1, further comprising a first dichroic mirror for collecting the fluorescence and transmitting the fluorescence to the first dichroic mirror, and an objective for reflecting the excitation light to the sample under test and for transmitting the fluorescence to the imaging assembly.
8. The imaging system of claim 7, wherein the imaging assembly comprises a plurality of second dichroic mirrors that distribute the fluorescence light to a plurality of imaging channels, the second dichroic mirrors being located between the imaging channels and the first dichroic mirrors, each of the imaging channels comprising a converging lens for converging the fluorescence light to the line camera and a line camera for receiving the fluorescence light.
9. An imaging method of an imaging system according to claim 1, wherein the imaging method comprises:
adjusting the first beam rotating component to a first state, and emitting excitation light through the light source component so as to irradiate the sample to be detected and excite fluorescence of a first line area extending along a first direction;
Receiving, by the imaging assembly, the first line area fluorescence to generate a first area array fluorescence image set of one area;
adjusting the first beam rotating component to a second state, and exciting fluorescence of a second line area extending along a second direction by irradiating the sample to be detected with emitted excitation light, wherein the second direction is perpendicular to the first direction;
receiving the second line area fluorescence again through the imaging assembly to generate a second area array fluorescence image set of one area;
and combining the super-resolution image according to the first area array fluorescent image group and the second area array fluorescent image group through the imaging component.
10. The imaging method of claim 9, wherein the first area array fluorescence image generation method comprises:
the objective table is driven by the driving component to move the sample to be detected along the second direction, meanwhile, the light source component is controlled to enable the excitation light to irradiate the sample to be detected along the second direction and excite a plurality of first line areas to emit fluorescence, at least one first line area which is not irradiated exists among the fluorescence of the first line areas, each first line area is excited to emit fluorescence, and the first area array fluorescence image of one area can be generated by the imaging component.
11. The imaging method of claim 9, wherein the method of generating the second area array fluorescence image comprises:
the object stage is driven to move the sample to be detected along the first direction through the driving component, meanwhile, the light source component is controlled to enable the excitation light to irradiate the sample to be detected along the first direction and excite a plurality of second line area fluorescence, at least one non-irradiated second line area exists between the second line area fluorescence, each time the second line area fluorescence is excited, and the second area array fluorescence image of one area can be generated through the imaging component.
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| KR20090020731A (en) * | 2007-08-24 | 2009-02-27 | 세크론 주식회사 | Apparatus for measuring the thickness of semiconductor device and resin molding system including the same |
| CN115372329A (en) * | 2022-08-29 | 2022-11-22 | 华南师范大学 | Near-infrared structured light illumination and polished section imaging method and device based on fluorescence loss super-resolution method |
| CN116718578A (en) * | 2023-08-11 | 2023-09-08 | 深圳赛陆医疗科技有限公司 | Super-resolution microscopic imaging system and imaging method |
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