Imaging system, sequencing system, and spatial protein component analysis system

Abstract

An imaging system, a sequencing system, and a spatial protein analysis system. The imaging system comprises: an excitation light source module (100) and an imaging module (200). The imaging module (200) comprises a filter set (210) and an image sensor (220). The filter set (210) comprises M filters (211), and the M filters (211) enter an imaging optical path in a preset order and transmit emitted light, allowing N types of emitted light having different wavelengths to sequentially enter a same image sensor (220) for imaging. Alternatively, the filter set (210) comprises K filters (211), and the N types of emitted light, after being split by a light-splitting module (300), pass through different filters (211) and enter different image sensors (220) for imaging. The imaging system can use the filters (211) to allow different emitted light to enter the same image sensor (220) in sequence, or use the filters (211) and the light-splitting module (300) to allow different emitted light to enter different image sensors (220), thereby suppressing signal crosstalk between emitted light having different wavelengths. The sequencing system comprises an imaging system. The spatial protein component analysis system comprises an imaging system.

Description

Imaging systems, sequencing systems, and spatial protein composition analysis systems Technical Field

[0001] The present application relates to the field of microscopic imaging technology, and in particular to an imaging system, a sequencing system, and a spatial protein component analysis system. Background Art

[0002] Studies have shown that the tumor microenvironment is a key factor affecting the effectiveness of immunotherapy. Systematically analyzing the tumor microenvironment and clarifying the related mechanisms of immunotherapy will provide a theoretical basis for relieving tumor microenvironment-mediated immunosuppression. It has great clinical value and scientific significance for precise immunotherapy of tumors, efficacy prediction, and medication guidance.

[0003] Fluorescence-encoded spatial proteomics, a recently developed technology, utilizes DNA-encoded fluorescence and cyclic excision staining to simultaneously image dozens of targets. It boasts not only rapid imaging (single-channel 4x4 mm² field of view, 0.6 μm / pixel, in approximately 8 minutes) but also high spatial resolution (up to 120 nm). Furthermore, two-photon microscopy enables in situ three-dimensional imaging of tissues with a high penetration depth (up to 700-1000 μm). These advantages make it ideal for in situ investigation of the microenvironment of entire tumor tissues. For example, Christian et al. reported using fluorescence-encoded CODEX technology to study the microenvironment of colorectal cancer, enabling simultaneous detection of 57 proteins and identifying nine cellular neighborhoods within the immune microenvironment and their interactions. Recently, Carole et al. reported using single-cell sequencing and in situ imaging of 23 protein markers using CODEX to delineate Tfr heterogeneity. They identified CD38, a key marker that characterizes the differences in Tfr cell origin, and also explained the functional determinants of Tfr cells within distinct structures of tonsillar germinal centers. Therefore, fluorescence-encoded spatial proteomics technology is expected to bring breakthroughs in the systematic analysis of the complex tumor microenvironment, and provide a theoretical basis and treatment plan for regulating the microenvironment to achieve immune suppression and clinical precision immunotherapy for malignant tumors.

[0004] However, fluorescent dyes have broad emission spectra, and there is some overlap between the emission spectra of different dyes, which can lead to crosstalk in the fluorescence signals and affect the accuracy of target encoding. Therefore, providing a fluorescence imaging system with low signal crosstalk is crucial for fluorescence-encoded spatial proteomics technology. Summary of the Invention

[0005] In view of this, the present application provides an imaging system, a sequencing system, and a spatial protein component analysis system, the scheme of which is as follows:

[0006] An imaging system comprising:

[0007] An excitation light source module, which emits excitation light beams of at least two different wavelengths to excite the sample to be tested to generate emission light of N different wavelengths;

[0008] An imaging module, comprising a filter set and an image sensor;

[0009] The filter set includes M filters, each of which allows the emission light of a corresponding wavelength to pass through and blocks the emission light of other wavelengths; the M filters enter the imaging light path of the imaging system in a preset order and pass the emission light of corresponding wavelengths, so that the emission light of N different wavelengths enters the same image sensor in sequence for imaging; or

[0010] The filter set includes K filters, each of which allows the emission light of one or more corresponding wavelengths to pass through and blocks the emission light of other wavelengths. The imaging system includes a spectroscopic module, and the emission light of N different wavelengths is split by the spectroscopic module, then passes through the corresponding filters and enters the corresponding image sensor for imaging.

[0011] Wherein, M=N and M, N≥2, 2≤K≤N, and M, N, and K are all integers.

[0012] In some embodiments, the imaging system further includes a lens module;

[0013] A light beam transmission module is provided between the excitation light source module and the lens module.

[0014] In some embodiments, the beam transmission module includes a first dichroic mirror and a second dichroic mirror, wherein the first dichroic mirror reflects the excitation beam to the second dichroic mirror;

[0015] The lens module has an optical axis, the second dichroic mirror is located on the optical axis of the lens module, the second dichroic mirror receives the excitation light beam from the first dichroic mirror and reflects the excitation light beam to the lens module; and

[0016] The dichroic mirror allows N different wavelengths of the emitted light to pass through and blocks other light beams from passing through.

[0017] In some embodiments, the imaging system further includes a focusing module and a driving module;

[0018] The focusing module includes a focusing light source, a focusing sensor, a processor and a driving module;

[0019] The focusing light source emits a focusing beam, and the focusing beam passes through the first dichroic mirror and is reflected by the second dichroic mirror to enter the lens module;

[0020] The focus sensor receives a focus beam reflected from the sample to be measured;

[0021] The processor determines, based on the focus light beam reflected from the sample to be tested and received by the focus sensor, a defocus amount of the sample to be tested relative to the lens module;

[0022] The driving module drives the lens module to move along its optical axis based on the defocus amount, so that the sample to be tested is located on the focal plane of the lens module and / or a clear image of the sample to be tested is obtained.

[0023] In some embodiments, a collimating mirror is disposed between the focusing module and the first dichroic mirror and / or between the excitation light source module and the first dichroic mirror.

[0024] In some embodiments, the filter set is located between the second dichroic mirror and the image sensor.

[0025] In some embodiments, a focusing lens is disposed between the filter set and the image sensor.

[0026] In some embodiments, the excitation light source module includes N excitation light sources;

[0027] The N excitation light sources are turned on in sequence according to a preset order, and each excitation light source excites the sample to be tested to generate emission light of one wavelength among N emission lights of different wavelengths.

[0028] In some embodiments, the excitation light source module includes a first excitation light source and a second excitation light source;

[0029] The first excitation light source emits a first excitation light beam;

[0030] The second excitation light beam emits a second excitation light beam;

[0031] The wavelength of the first excitation light beam is different from the wavelength of the second excitation light beam.

[0032] In some embodiments, the first excitation light source and the second excitation light source emit the first excitation light beam and the second excitation light beam in a time-sharing manner;

[0033] The first excitation light beam excites the sample to be tested to generate NL different wavelengths of emission light among the N different wavelengths of emission light; the second excitation light beam excites the sample to be tested to generate the remaining wavelengths of emission light;

[0034] Wherein, 1≤L<N, and L is an integer.

[0035] In some embodiments, the imaging system includes a turntable, and the M filters are arranged on the turntable. When the turntable is rotated, the M filters enter the imaging light path of the imaging system in a preset order.

[0036] In some embodiments, the first excitation light beam and the second excitation light beam excite the sample to be tested in a time-sharing manner to generate emission light of five different wavelengths;

[0037] The first excitation light beam excites the sample to be tested to generate emission light of a first wavelength, emission light of a second wavelength, and emission light of a third wavelength, and the second excitation light beam excites the sample to be tested to generate emission light of a fourth wavelength and emission light of a fifth wavelength;

[0038] The image sensor includes a first image sensor, a second image sensor and a third image sensor;

[0039] The optical splitting module includes:

[0040] a third dichroic mirror, the third dichroic mirror being opposite to the light-emitting surface of the second dichroic mirror, transmitting the emission light of the first wavelength and the emission light of the fourth wavelength to the first image sensor, and reflecting the remaining fluorescence;

[0041] a fourth dichroic mirror, opposite to a reflective surface of the third dichroic mirror, reflecting the emission light of the second wavelength and the emission light of the fifth wavelength to the second image sensor, and transmitting the emission light of the third wavelength to the third image sensor.

[0042] In some embodiments, the filter set includes a first filter, a second filter, and a third filter;

[0043] The first filter is located between the third dichroic mirror and the first image sensor, the first filter is opposite to the light-exiting surface of the third dichroic mirror, and is used to filter the emission light of the first wavelength and the emission light of the fourth wavelength. The first image sensor is located on one side of the light-exiting surface of the first filter;

[0044] The second filter is located between the fourth dichroic mirror and the second image sensor, the second filter is opposite to the reflective surface of the fourth dichroic mirror, and is used to filter the emission light of the second wavelength and the emission light of the fifth wavelength, and the second image sensor is located on the light exiting surface side of the second filter;

[0045] The third filter is located between the fourth dichroic mirror and the third image sensor. The third filter is opposite to the light-transmitting surface of the fourth dichroic mirror and is used to filter the emitted light of the third wavelength. The third image sensor is located on the light-emitting side of the third filter.

[0046] A sequencing system comprises the imaging system described in any one of the above embodiments.

[0047] A spatial protein component analysis system comprises the imaging system described in any one of the above embodiments.

[0048] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0049] The imaging system provided by the present application includes: an excitation light source module and an imaging module, wherein the imaging module includes a filter set and an image sensor. The filter set includes M filters, each of which allows emission light of a corresponding wavelength to pass through and blocks emission light of other wavelengths. The M filters enter the imaging light path of the imaging system in a preset order and pass emission light of corresponding wavelengths, so that emission light of N different wavelengths enters the same image sensor in sequence for imaging; or the filter set includes K filters, each of which allows emission light of one or more corresponding wavelengths to pass through and blocks emission light of other wavelengths. The imaging system also includes a spectrometer module, and the emission light of N different wavelengths is split by the spectrometer module, passes through corresponding filters, and enters the corresponding image sensor for imaging. It can be seen from this that the imaging system can use filters to allow emission light of different wavelengths to enter the same image sensor in sequence, or use filters and spectrometer modules to allow emission light of different wavelengths to enter corresponding different image sensors. Then, each wavelength of emission light has a corresponding image sensor, that is, each wavelength of emission light is imaged separately, thereby suppressing the signal crosstalk between emission light of different wavelengths, making the fluorescence signal crosstalk of the imaging system lower, and improving the imaging quality of the sample to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or related technologies, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without any creative work.

[0051] The structures, proportions, sizes, etc. depicted in the drawings of this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not intended to limit the conditions under which this application can be implemented, and therefore have no substantive technical significance. Any structural modifications, changes in proportional relationships, or adjustments in size, without affecting the efficacy and objectives that can be achieved by this application, should still fall within the scope of the technical contents disclosed in this application.

[0052] Figure 1 is a fluorescence spectrum diagram of different emission lights;

[0053] FIG2 is a schematic structural diagram of an imaging system provided by the present application;

[0054] FIG3 is a schematic structural diagram of another imaging system provided by the present application;

[0055] FIG4 is a schematic structural diagram of another imaging system provided by the present application;

[0056] FIG5 is a schematic structural diagram of another imaging system provided by the present application;

[0057] FIG6 is a schematic structural diagram of another imaging system provided in this application. DETAILED DESCRIPTION

[0058] The following will be combined with the accompanying drawings to clearly and completely describe the embodiments of this application. Obviously, the described embodiments are only embodiments of one area of ​​this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0059] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0060] As described in the background technology section, due to the wide emission spectra of fluorescent dyes, the emission spectra of different dyes may overlap, as shown in Figure 1. This will cause crosstalk between the signals of different fluorescent dyes, causing the imaging of different fluorescent dyes to affect each other, resulting in poor imaging quality and affecting the accuracy of target coding.

[0061] Based on the above, the present application provides an imaging system, which includes:

[0062] The excitation light source module 100 emits at least two excitation light beams of different wavelengths to excite the sample to be tested to produce N emission lights of different wavelengths, that is, the excitation light beam emitted by the excitation light source module 100 can laser the sample to be tested to produce emission light, thereby producing N emission lights of different wavelengths.

[0063] The imaging module 200 includes a filter group 210 and an image sensor 220. The filter group 210 is used to filter emission lights of N different wavelengths, and the image sensor 220 is used to image the sample to be tested based on the emission lights of N different wavelengths.

[0064] Based on the above, in one embodiment of the present application, as shown in FIG3 , a filter set 210 includes M filters 211 , each filter 211 allowing emission light of a corresponding wavelength to pass through and blocking emission light of other wavelengths. For example, if the N different wavelengths of emission light are A, B, C, D, and E, the first filter of the M filters 211 allows emission light A to pass through while blocking emission light B, C, D, and E from continuing to pass through it. The second filter allows emission light B to pass through while blocking emission light A, C, D, and E from continuing to pass through it, and so on. The M filters 211 enter the imaging optical path of the imaging system in a preset order and transmit emission light of corresponding wavelengths, so that the emission light of N different wavelengths enters the same image sensor 220 in sequence. Wherein, M=N, and M, N≥2, and M, N are both integers.

[0065] As can be seen from the above, in this embodiment, when the imaging system is working, the M filters 211 will enter the imaging light path in sequence, and each filter 211 only allows the corresponding emission light of one wavelength to pass through. As the M filters 211 enter the imaging light path in sequence, the N emission lights of different wavelengths can enter the image sensor 220 in sequence to image the sample to be tested. Since the M filters 211 enter the imaging light path in sequence, the emission lights of N different wavelengths will also enter the image sensor 220 in sequence. Then, the emission lights of N different wavelengths will not enter the imaging light path at the same time, and thus will not enter the same image sensor 220 at the same time. That is, the emission lights of N different wavelengths will not simultaneously image the sample to be tested. Therefore, in theory, there is no signal crosstalk between the emission lights of different wavelengths. This means that the imaging system can effectively suppress the signal crosstalk between the emission lights of different wavelengths, so that the fluorescence signal crosstalk of the imaging system is low, and has better imaging effect, which is of great significance for fluorescence-encoded spatial proteomics technology.

[0066] In another embodiment of the present application, as shown in FIG2 , a filter set 210 includes K filters 211 , each of which allows emission light of one or more corresponding wavelengths to pass through while blocking emission light of other wavelengths. Based on this, the imaging system further includes a spectrometer module 300 . After being split by the spectrometer module 300 , the N different wavelengths of emission light pass through corresponding filters 211 and enter corresponding image sensors 220 for imaging. That is, each wavelength of emission light enters a corresponding image sensor 220 for imaging. Where 2 ≤ K ≤ N, and N and K are both integers. Based on the description of this embodiment, it can be seen that the difference between this embodiment and the above embodiments is that: this embodiment has an image sensor 220 that corresponds one-to-one to N kinds of emission light of different wavelengths. After the N kinds of emission light of different wavelengths are filtered by the filter 211 and split by the spectrometer module 300 in turn, they can enter the corresponding image sensor 220 for imaging respectively, so there is no crosstalk problem between emission lights of different wavelengths. Therefore, the imaging system can effectively suppress the signal crosstalk between emission lights of different wavelengths, so that the fluorescence signal crosstalk of the imaging system is low.

[0067] In summary, the imaging system provided by the present application can use filters to allow emission light of different wavelengths to enter the same image sensor in sequence, or use filters and spectrometer modules to allow emission light of different wavelengths to enter corresponding different image sensors, so that each wavelength of emission light has a corresponding image sensor, that is, each wavelength of emission light is imaged separately, which can suppress the signal crosstalk between emission light of different wavelengths, so that the fluorescence signal crosstalk of the imaging system is low, and the imaging quality of the sample to be tested can be improved.

[0068] Based on the above, in one embodiment of the present application, the imaging system also includes a lens module 400, for example, the lens module 400 includes an objective lens, etc., and a light beam transmission module 500 is arranged between the excitation light source module 100 and the lens module 400, so that the excitation light beam emitted by the excitation light source module 100 can be transmitted to the sample to be tested through the light beam transmission module 500 to excite the sample to be tested to generate N different wavelengths of emission light.

[0069] Based on the above embodiment, in one embodiment of the present application, the light beam transmission module 500 includes a first dichroic mirror 510 and a second dichroic mirror 520. The first dichroic mirror 510 reflects the excitation light beam to the second dichroic mirror 520, that is, the second dichroic mirror 520 is located on the reflective surface of the first dichroic mirror 510. The lens module 400 has an optical axis, and the second dichroic mirror 520 is located on the optical axis of the lens module 400. The lens module 400 is located on the reflective surface of the second dichroic mirror 520. The second dichroic mirror 520 receives the excitation light beam from the first dichroic mirror 510 and reflects the excitation light beam transmitted through the first dichroic mirror 510 to the lens module 400. The excitation light beam is then converged onto the sample to be tested through the lens module 400, thereby exciting the sample to be tested to generate emission light of N different wavelengths.

[0070] Based on the above, it can be seen that when the imaging system is working, the excitation light source module 100 emits an excitation light beam. After the excitation light beam is transmitted to the first dichroic mirror 510, it is reflected by the first dichroic mirror 510 to the second dichroic mirror 520, and then reflected by the second dichroic mirror 520 to the lens module 400. Therefore, the light beam transmission module 400 can transmit the excitation light beam emitted by the excitation light source module 100 to the lens module 400, and then transmit it to the sample to be tested through the lens module 400, thereby exciting the sample to be tested to generate N different wavelengths of emission light.

[0071] In addition, in this embodiment, the second dichroic mirror 520 also allows N different wavelengths of emission light to pass through and prevents other light beams from passing through. That is, the second dichroic mirror 520 can allow N different wavelengths of emission light to pass through, but prevents light beams other than the emission light from continuing to transmit through it. Therefore, after the excitation light beam excites the sample to be tested to generate N different wavelengths of emission light, the second dichroic mirror 520 can prevent light beams other than the above-mentioned N different wavelengths of emission light from entering the imaging light path, thereby suppressing signal crosstalk between the excitation light beam and the emission light beam, which helps to improve the imaging quality of the imaging system.

[0072] On the basis of the above embodiment, in one embodiment of the present application, as shown in FIG4 , the imaging system further includes a focusing module 600 and a driving module 700 , and the specific working process is as follows:

[0073] The focusing module 600 includes a focusing light source 610, a focusing sensor 620, a processor 630 and a driving module 700. The focusing light source 610 emits a focusing beam. The focusing light source 610 is located on the light-transmitting surface side of the first dichroic mirror 510. The focusing beam passes through the first dichroic mirror 510 and then enters the lens module 400 through the second dichroic mirror 520.

[0074] The focus sensor 620 receives the focus beam reflected from the sample to be measured.

[0075] The processor 630 determines the defocus amount of the sample to be tested relative to the lens module 400 based on the focus light beam reflected from the sample to be tested and received by the focus sensor 620. The defocus amount is the distance between the plane where the sample to be tested is located or the surface of the sample to be tested and the focal plane of the lens module 400.

[0076] The driving module 700 drives the lens module 400 to move along its optical axis based on the defocus amount so that the sample to be tested is located on the focal plane of the lens module 400 and / or a clear image of the sample to be tested is obtained. That is, the driving module 700 drives the lens module 400 to move along its optical axis so that the sample to be tested is located on the focal plane of the lens module 400, or the driving module 700 drives the lens module 400 to move along its optical axis to obtain a clear image of the sample to be tested, or the driving module 700 drives the lens module 400 to move along its optical axis so that the sample to be tested is located on the focal plane of the lens module 400 and a clear image of the sample to be tested is obtained at the same time. It should be noted that, although this embodiment describes that the driving module 700 drives the lens module 400 to move along its optical axis so that the sample to be tested is located on the focal plane of the lens module 400 and / or a clear image of the sample to be tested is obtained, in actual applications, the driving module 700 can also be used to drive the sample to be tested to move along the optical axis of the lens module 400 so that the sample to be tested is located on the focal plane of the lens module 400 and / or a clear image of the sample to be tested is obtained, depending on the specific circumstances.

[0077] Based on the above, when the imaging system is focused, the focusing light source 610 generates and emits a focusing beam. The focusing beam passes through the first dichroic mirror 510 and the second dichroic mirror 520 in sequence, and is then transmitted to the sample to be tested by the lens module 400. After being reflected by the sample to be tested, it is received by the focusing sensor 620. Then, the processor 630 will use the focusing beam reflected from the sample to be tested received by the focusing sensor 620 to determine the defocus amount of the sample to be tested relative to the lens module 400. After obtaining the defocus amount, the driving module 700 will drive the lens module 400 and / or the sample to be tested to move to eliminate the defocus amount.

[0078] Based on the above embodiments, in one embodiment of the present application, as shown in Figure 5, the imaging system also includes a collimator, and a collimator is arranged between the focusing module 600 and the first dichroic mirror 510 and / or a collimator is arranged between the excitation light source module 100 and the first dichroic mirror 510. For example, the collimator may include a first collimator 650 and / or a second collimator 660, and a first collimator 620 may be arranged between the focusing module 600 and the first dichroic mirror 510 to collimate the focusing beam and improve the directionality of the collimated beam, or a second collimator 660 may be arranged between the excitation light source module 100 and the first dichroic mirror 510 to collimate the excitation beam and improve the directionality of the excitation beam, or a first collimator 650 may be arranged between the focusing module 600 and the first dichroic mirror 510, and a second collimator 660 may be arranged between the excitation light source module 100 and the first dichroic mirror 510 to collimate both the focusing beam and the excitation beam and improve the directionality of both, so as to help ensure the imaging quality of the imaging system.

[0079] Based on the above embodiment, the filter set 210 is positioned between the second dichroic mirror 520 and the image sensor 220, so that the emission light transmitted through the second dichroic mirror 520 can be transmitted to the image sensor 220 through the filter set 210 for imaging. For example, emission light of N different wavelengths allowed by the second dichroic mirror 520 and further transmitted through the second dichroic mirror 520 passes through the corresponding filters 211 and enters the same image sensor 220 for imaging. Alternatively, emission light of N different wavelengths allowed by the second dichroic mirror 520 and further transmitted through the second dichroic mirror 520 passes through the filters 211 and the spectrometer module 300 and enters N image sensors for imaging.

[0080] Based on the above embodiments, in one embodiment of the present application, a focusing lens 700 is provided between the filter group 210 and the image sensor 220 to focus the emission light transmitted through the filter 211 in the filter group 210 to the image sensor 220 for imaging.

[0081] On the basis of the above embodiments, in one embodiment of the present application, for the case where N kinds of emission lights of different wavelengths correspond one-to-one to the image sensor, as shown in FIG5 , the excitation light source module 100 includes N excitation light sources 110. During the operation of the imaging system, the N excitation light sources 110 are turned on in sequence according to a preset order, and each of the N excitation light sources 110 excites the sample to be tested to generate emission light of one wavelength among the N kinds of emission lights of different wavelengths, that is, the N excitation light sources 110 correspond one-to-one to the N kinds of emission lights of different wavelengths. Therefore, during the operation of the imaging system, the N excitation light sources 110 can be turned on in sequence according to a preset order, and can also excite the sample to be tested to generate N kinds of emission lights of different wavelengths one by one in sequence, so that the light beams with overlapping spectra in the emission light will not enter the imaging optical path at the same time, and thus will not enter the image sensor 220 at the same time, thereby reducing the signal crosstalk between the emission lights of different wavelengths and improving the imaging quality of the imaging system. It should be noted that, since emission light of different wavelengths can be excited by excitation light beams with the same or similar wavelengths, the wavelengths of the N excitation light beams emitted by the above-mentioned N excitation light sources can be different, or the wavelengths of some of the N excitation light beams can be the same, depending on the wavelengths of the excitation light beams corresponding to the emission light of N different wavelengths.

[0082] In another embodiment of the present application, as shown in FIG6 , the excitation light source module 100 includes a first excitation light source 120 and a second excitation light source 130. The first excitation light source 120 is configured to emit a first excitation light beam, and the second excitation light source 130 is configured to emit a second excitation light beam. The wavelength of the first excitation light beam is different from the wavelength of the second excitation light beam.

[0083] According to the description of this embodiment, it can be seen that the difference between this embodiment and the above embodiment is that the excitation light source module 100 in the above embodiment has an excitation light source corresponding to N emission lights of different wavelengths, that is, the excitation light source module 100 in the above embodiment has N excitation light sources 110. However, in actual applications, emission lights of different wavelengths can be excited by an excitation light beam of the same wavelength. Therefore, in this embodiment, the excitation light source module 100 can include two excitation light sources, namely a first excitation light source 120 and a second excitation light source 130. In other words, the excitation light source module 100 can include less excitation light sources than the number of emission lights, thereby reducing the number of excitation light sources in the excitation light source module 100, thereby reducing the volume of the excitation light source module 100 and the cost of the excitation light source module 100, thereby reducing the volume and cost of the imaging system. It should be noted that in this embodiment, the excitation light source module 100 includes two excitation light sources, which is only an example. According to actual conditions, on the basis that the number of excitation light sources is less than the number of emission lights, the number of excitation light sources in the excitation light source module can also be other values, depending on the specific situation.

[0084] On the basis of the above embodiments, in one embodiment of the present application, the first excitation light source 120 and the first excitation light source 130 emit the first excitation light beam and the second excitation light beam at different times, that is, the first excitation light beam and the second excitation light beam are not emitted at the same time, but are emitted in sequence, for example, the first excitation light source 120 first emits the first excitation light beam, and then the second excitation light source 130 emits the second excitation light beam, or the second excitation light source 130 first emits the second excitation light beam, and then the first excitation light source 120 emits the first excitation light beam.

[0085] Furthermore, the first excitation light beam excites the sample to be tested to generate NL kinds of emission light of different wavelengths among the N kinds of emission light of different wavelengths, and the second excitation light beam excites the sample to be tested to generate emission light of the remaining wavelengths, where 1≤L<N, and L is an integer. In other words, the first excitation light beam emitted by the first excitation light source 120 can excite the sample to be tested to generate emission light of some wavelengths, and the second excitation light beam emitted by the second excitation light source 130 can excite the sample to be tested to generate emission light of the remaining part, so that the first excitation light source 120 and the first excitation light source 130 emit the first excitation light beam and the second excitation light beam in a time-sharing manner, and can excite the sample to be tested in a time-sharing manner to successively generate emission light of N different wavelengths, perform imaging of the sample to be tested, suppress mutual interference between emission light of different wavelengths, and improve the imaging quality of the sample to be tested.

[0086] Based on the fact that the filter group 210 includes M filters 211, in one embodiment of the present application, the imaging system includes a turntable 2111, and the M filters 211 are set on the turntable 2111. By rotating the turntable 2111, the M filters can enter the imaging light path of the imaging system in sequence, and can pass the emission light of corresponding wavelengths among the N different wavelengths of emission light, and block the emission light of other wavelengths, thereby achieving the isolation of N different wavelengths of emission light, suppressing the interference between the N different wavelengths of emission light, and ensuring the imaging quality of the sample to be tested.

[0087] Taking the example of an excitation light beam exciting the sample to be tested to generate emission light of five different wavelengths, they are respectively recorded as emission light of a first wavelength, emission light of a second wavelength, emission light of a third wavelength, emission light of a fourth wavelength, and emission light of a fifth wavelength. Based on the fact that the filter group 210 includes K filters 211, in one embodiment of the present application, the first excitation light beam and the second excitation light beam excite the sample to be tested in a time-sharing manner, causing the sample to be tested to generate emission light of five different wavelengths. For example, the first excitation light beam can excite the sample to be tested to generate emission light of a first wavelength, emission light of a second wavelength, and emission light of a third wavelength, and the second excitation light beam excites the sample to be tested to generate emission light of a fourth wavelength and emission light of a fifth wavelength.

[0088] Based on the above, the image sensor 220 includes a first image sensor 221 , a second image sensor 222 , and a third image sensor 223 , and the light splitting module 300 includes a third dichroic mirror 310 and a fourth dichroic mirror 320 .

[0089] The third dichroic mirror 310 is opposite to the light emitting surface of the second dichroic mirror 520 , transmits the emission light of the first wavelength and the emission light of the fourth wavelength to the first image sensor 211 , and blocks the emission light of other wavelengths.

[0090] Fourth dichroic mirror 320 is opposite to the reflective surface of third dichroic mirror 310 , reflects the emission light of the second wavelength and the emission light of the fifth wavelength to second image sensor 222 , and transmits the emission light of the third wavelength to third image sensor 223 .

[0091] Based on the above, the first excitation light source 120 emits a first excitation light beam to excite the sample to be tested to generate emission light of the first wavelength, emission light of the second excitation wavelength, and emission light of the third excitation wavelength. The emission light of the three wavelengths is then transmitted to the third dichroic mirror 310 through the second dichroic mirror 520. Because the third dichroic mirror 310 only transmits the emission light of the first wavelength and the emission light of the fourth wavelength, when the emission light of the three wavelengths is transmitted to the third dichroic mirror 310, only the emission light of the first wavelength is transmitted to the first image sensor for imaging, and the emission light of the second wavelength and the emission light of the third wavelength are reflected to the fourth dichroic mirror 320. Since the fourth dichroic mirror 320 only transmits emission light of the third wavelength, the fourth dichroic mirror 320 can reflect the emission light of the second wavelength transmitted thereto to the second image sensor 222 for imaging, and transmit the emission light of the third wavelength to the third image sensor 223 for imaging, so that the emission light of the first wavelength, the emission light of the second wavelength, and the emission light of the third wavelength that are excited at the same time enter different image sensors for imaging, respectively, thereby avoiding signal crosstalk between emission light of different wavelengths, and improving the imaging quality of the sample to be tested.

[0092] Similarly, the second excitation light source 130 emits a second excitation light beam to excite the sample to be tested to generate emission light of a fourth wavelength and emission light of a fifth wavelength. When the emission light of the fourth wavelength and the emission light of the fifth wavelength are transmitted to the third dichroic mirror 310 through the lens module 400, the emission light of the fourth wavelength will be transmitted through the third dichroic mirror 310 to the first image sensor for imaging, while the emission light of the fifth wavelength will be reflected by the third dichroic mirror 320 to the fourth dichroic mirror 320, and reflected by the fourth dichroic mirror 320 to the second image sensor 222 for imaging, so that the emission light of the fourth wavelength and the emission light of the fifth wavelength generated at the same time enter different image sensors, suppressing the interference between the emission lights of different wavelengths, and improving the imaging quality of the sample to be tested.

[0093] Based on the above embodiment, in one embodiment of the present application, as shown in FIG6 , the filter set 210 includes a first filter 212, a second filter 213, and a third filter 214. The first filter 212 is located between the third dichroic mirror 310 and the first image sensor 221, and the first filter 212 is opposite the light-exiting surface of the third dichroic mirror 310. The filter set 210 is configured to filter the emission light of the first wavelength and the emission light of the fourth wavelength. The first image sensor 221 is located on the light-exiting surface of the first filter 212. Therefore, the emission light of the first wavelength and the emission light of the fourth wavelength transmitted through the third dichroic mirror 310 are filtered by the first filter before entering the first image sensor 221. This effectively prevents light beams other than the emission light of the first wavelength and the emission light of the fourth wavelength from entering the first image sensor 221, thereby reducing the impact of the remaining light beams on the imaging of the emission light of the first wavelength and the emission light of the fourth wavelength, thereby improving the imaging quality of the sample to be measured.

[0094] The second filter 213 is located between the fourth dichroic mirror 320 and the second image sensor 222. The second filter 213 faces the reflective surface of the fourth dichroic mirror 320 and is configured to filter the emission light of the second wavelength and the emission light of the fifth wavelength. The second image sensor 222 is located on one side of the light-exiting surface of the second filter 213. Because the second filter 213 faces the reflective surface of the fourth dichroic mirror 320, the emission light of the second wavelength and the emission light of the fifth wavelength reflected by the fourth dichroic mirror 320 are transmitted to the second filter 213 and filtered by the second filter 213 before entering the second image sensor 222. This effectively prevents light beams other than the emission light of the second wavelength and the emission light of the fifth wavelength from entering the second image sensor 222, thereby reducing the impact of the remaining light beams on the imaging of the emission light of the second wavelength and the emission light of the fifth wavelength, thereby improving the imaging quality of the sample to be tested.

[0095] The third filter 214 is located between the fourth dichroic mirror 320 and the third image sensor 223. The third filter 214 and the light-transmitting surface of the fourth dichroic mirror 320 are opposite to each other and are used to filter the emission light of the third wavelength. The third image sensor 223 is located on the light-emitting side of the third filter 214. Therefore, the emission light of the third wavelength transmitted through the fourth dichroic mirror 320 will be filtered by the third filter 214 before entering the third image sensor 223, thereby reducing the influence of other light beams on the imaging of the emission light of the third wavelength, thereby improving the imaging quality of the sample to be measured.

[0096] Based on the imaging system described in any of the above embodiments, in a specific embodiment of the present application, the excitation light source module of the imaging system includes three excitation light sources, the wavelengths of the excitation light beams are 488nm, 532nm, and 660nm respectively, and the N different wavelengths of emission light are five kinds of emission light generated by the five fluorescent dyes Sytox Green, ATTO532, ROX, CY5, and IF700 excited by the three excitation light beams, with wavelengths of 512nm, 551nm, 602nm, 670nm, and 712nm respectively. The scanning range of the imaging system can be 20mm×20mm, the imaging mode can be wide-field fluorescence imaging, the spatial resolution can be ≤600nm, the magnification can be 12.8, the image size can be 4112 pixels×2176 pixels, the object field of view can be 1.10mm×0.58mm, the imaging speed can be ≤400ms / field of view, the stage field of view switching time can be ≤80ms / field of view, and the stage positioning accuracy can be ≤200nm. The sample to be tested is placed on the stage, and the sample to be tested can be paraffin-embedded or frozen tissue sections or nucleic acid samples, etc.

[0097] Based on the imaging system described in any of the above embodiments, the present application further provides a sequencing system, comprising the imaging system described in any of the above embodiments. The purpose of including the imaging system in the sequencing system is to suppress crosstalk between emitted light of different wavelengths during operation of the sequencing system, thereby ensuring the imaging quality of images used for sequencing analysis and thereby improving the accuracy of the sequencing system.

[0098] Based on the imaging system described in any of the above embodiments, the present application also provides a spatial protein analysis system, which includes the imaging system described in any of the above embodiments. Since the imaging system can suppress the crosstalk problem between emission lights of different wavelengths, that is, it can suppress the crosstalk problem of fluorescence signals during spatial protein analysis, it helps to ensure the accuracy of spatial protein analysis.

[0099] It should be noted that the above-mentioned sequencing system and spatial protein analysis system have improved the structure of their imaging systems and suppressed the problem of fluorescence signal crosstalk during the imaging process, but the remaining working processes of the sequencing system and the spatial protein analysis system, such as sequencing, will not change. Therefore, this application will not go into details about the specific working process of the sequencing system.

[0100] In summary, the present application provides an imaging system, a sequencing system and a spatial protein separation system, the imaging system comprising: an excitation light source module and an imaging module, the imaging module comprising a filter set and an image sensor. The filter set comprises M filters, each of which allows emission light of a corresponding wavelength to pass through and blocks emission light of other wavelengths, the M filters enter the imaging light path of the imaging system in a preset order and pass emission light of corresponding wavelengths, so that emission light of N different wavelengths enters the same image sensor in sequence for imaging. Or the filter set comprises K filters, each of which allows emission light of one or more corresponding wavelengths to pass through and blocks emission light of other wavelengths, the imaging system further comprising a spectroscopic module, and emission light of N different wavelengths is spectroscopically split by the spectroscopic module and then passes through the corresponding filter and enters the corresponding image sensor for imaging. It can be seen from this that the imaging system can use filters to allow emission light of different wavelengths to enter the same image sensor in sequence, or use filters and spectrometer modules to allow emission light of different wavelengths to enter corresponding different image sensors. Then, each wavelength of emission light has a corresponding image sensor, that is, each wavelength of emission light is imaged separately, thereby suppressing the signal crosstalk between emission light of different wavelengths, making the fluorescence signal crosstalk of the imaging system lower, and improving the imaging quality of the sample to be tested.

[0101] The various embodiments in this specification are described in a progressive, parallel, or combined manner. Each embodiment focuses on the differences from other embodiments, and reference can be made to the descriptions of the embodiments for similar or identical areas. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the descriptions are relatively simple. For relevant details, refer to the descriptions of the methods.

[0102] It should be noted that in the description of this application, it should be understood that the terms "upper", "lower", "top", "bottom", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as limiting this application. When a component is considered to be "connected" to another component, it can be directly connected to the other component or there may be a centrally located component.

[0103] It should also be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such article or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the article or device comprising the aforementioned elements.

[0104] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present application. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An imaging system, characterized in that: include: An excitation light source module, which emits excitation light beams of at least two different wavelengths to excite the sample to be tested to generate emission light of N different wavelengths; An imaging module, comprising a filter set and an image sensor; The filter set includes M filters, each of which allows the emission light of a corresponding wavelength to pass through and blocks the emission light of other wavelengths; the M filters enter the imaging light path of the imaging system in a preset order and pass the emission light of corresponding wavelengths, so that the emission light of N different wavelengths enters the same image sensor in sequence for imaging; or The filter set includes K filters, each of which allows the emission light of one or more corresponding wavelengths to pass through and blocks the emission light of other wavelengths. The imaging system includes a spectroscopic module, and the emission light of N different wavelengths is split by the spectroscopic module, then passes through the corresponding filters and enters the corresponding image sensor for imaging. Wherein, M=N and M, N≥2, 2≤K≤N, and M, N, and K are all integers.

2. The imaging system according to claim 1, wherein: The imaging system further includes a lens module; A light beam transmission module is provided between the excitation light source module and the lens module.

3. The imaging system according to claim 2, wherein: The beam transmission module includes a first dichroic mirror and a second dichroic mirror, wherein the first dichroic mirror reflects the excitation beam to the second dichroic mirror; The lens module has an optical axis, the second dichroic mirror is located on the optical axis of the lens module, the second dichroic mirror receives the excitation light beam from the first dichroic mirror and reflects the excitation light beam to the lens module; and The dichroic mirror allows N different wavelengths of the emitted light to pass through and blocks other light beams from passing through.

4. The imaging system according to claim 3, wherein: The imaging system further includes a focusing module and a driving module; The focusing module includes a focusing light source, a focusing sensor, a processor and a driving module; The focusing light source emits a focusing beam, and the focusing beam passes through the first dichroic mirror and is reflected by the second dichroic mirror to enter the lens module; The focus sensor receives a focus beam reflected from the sample to be measured; The processor determines, based on the focus light beam reflected from the sample to be tested and received by the focus sensor, a defocus amount of the sample to be tested relative to the lens module; The driving module drives the lens module to move along its optical axis based on the defocus amount, so that the sample to be tested is located on the focal plane of the lens module and / or a clear image of the sample to be tested is obtained.

5. The imaging system according to claim 4, wherein: A collimating mirror is provided between the focusing module and the first dichroic mirror and / or between the excitation light source module and the first dichroic mirror.

6. The imaging system according to any one of claims 3 to 5, characterized in that: The filter set is located between the second dichroic mirror and the image sensor.

7. The imaging system according to claim 6, wherein: A focusing lens is arranged between the filter group and the image sensor.

8. The imaging system according to claim 6 or 7, characterized in that The excitation light source module includes N excitation light sources; The N excitation light sources are turned on in sequence according to a preset order, and each excitation light source excites the sample to be tested to generate emission light of one wavelength among N emission lights of different wavelengths.

9. The imaging system according to any one of claims 3 to 7, wherein: The excitation light source module includes a first excitation light source and a second excitation light source; The first excitation light source emits a first excitation light beam; The second excitation light beam emits a second excitation light beam; The wavelength of the first excitation light beam is different from the wavelength of the second excitation light beam.

10. The imaging system according to claim 9, wherein: The first excitation light source and the second excitation light source emit the first excitation light beam and the second excitation light beam in a time-sharing manner; The first excitation light beam excites the sample to be tested to generate NL types of emission lights of different wavelengths among N types of emission lights of different wavelengths; The second excitation light beam excites the sample to be tested to generate emission light of other wavelengths; Wherein, 1≤L<N, and L is an integer.

11. The imaging system according to claim 9 or 10, characterized in that The imaging system includes a turntable, and M filters are arranged on the turntable. When the turntable is rotated, the M filters enter the imaging light path of the imaging system in a preset order.

12. The imaging system according to claim 9 or 10, characterized in that The first excitation light beam and the second excitation light beam excite the sample to be tested in a time-sharing manner to generate emission light of five different wavelengths; The first excitation light beam excites the sample to be tested to generate emission light of a first wavelength, emission light of a second wavelength, and emission light of a third wavelength, and the second excitation light beam excites the sample to be tested to generate emission light of a fourth wavelength and emission light of a fifth wavelength; The image sensor includes a first image sensor, a second image sensor and a third image sensor; The optical splitting module includes: a third dichroic mirror, the third dichroic mirror being opposite to the light-emitting surface of the second dichroic mirror, transmitting the emission light of the first wavelength and the emission light of the fourth wavelength to the first image sensor, and reflecting the remaining fluorescence; a fourth dichroic mirror, opposite to a reflective surface of the third dichroic mirror, reflecting the emission light of the second wavelength and the emission light of the fifth wavelength to the second image sensor, and transmitting the emission light of the third wavelength to the third image sensor.

13. The imaging system according to claim 12, wherein: The filter set includes a first filter, a second filter and a third filter; The first filter is located between the third dichroic mirror and the first image sensor, the first filter is opposite to the light-exiting surface of the third dichroic mirror, and is used to filter the emission light of the first wavelength and the emission light of the fourth wavelength. The first image sensor is located on one side of the light-exiting surface of the first filter; The second filter is located between the fourth dichroic mirror and the second image sensor, the second filter is opposite to the reflective surface of the fourth dichroic mirror, and is used to filter the emission light of the second wavelength and the emission light of the fifth wavelength, and the second image sensor is located on the light exiting surface side of the second filter; The third filter is located between the fourth dichroic mirror and the third image sensor. The third filter is opposite to the light-transmitting surface of the fourth dichroic mirror and is used to filter the emitted light of the third wavelength. The third image sensor is located on the light-emitting side of the third filter.

14. A sequencing system comprising the imaging system according to any one of claims 1 to 13.

15. A spatial protein component analysis system comprising the imaging system according to any one of claims 1 to 13.

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