Imaging system, sequencing system and spatial protein component analysis system

Abstract

The utility model provides an imaging system, a sequencing system and a space protein component analysis system, and relates to the technical field of microscopic imaging, the imaging system comprises an excitation light source module and an imaging module, and the imaging module comprises an optical filter group and an image sensor. The optical filter group comprises M optical filters, and the M optical filters enter the imaging light path according to a preset sequence and penetrate through the corresponding emitted light, so that the N kinds of emitted light with different wavelengths sequentially enter the same image sensor for imaging. Or the optical filter group comprises K optical filters, and the N different emitted lights are split by the light splitting module, penetrate through the corresponding optical filters and enter the corresponding image sensors for imaging. According to the imaging system, different emitted light can enter the same image sensor in sequence through the optical filter, or the different emitted light can enter the corresponding different image sensors through the optical filter and the light splitting module, so that signal crosstalk between the emitted light with different wavelengths can be inhibited.

Description

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. Systematic analysis of the tumor microenvironment and clarification of immunotherapy-related mechanisms will provide a theoretical basis for relieving tumor microenvironment-mediated immunosuppression, which has great clinical value and scientific significance for precise immunotherapy of tumors, efficacy prediction, and medication guidance.

[0003] The spatial proteomics technology based on fluorescence coding, which has been developed in recent years, can simultaneously image dozens of targets by means of DNA-encoded fluorescence and circular excision staining. It not only has fast imaging speed (single-channel 4×4mm2 field of view, 0.6μm / pixel, only about 8 minutes) and high spatial resolution (up to 120nm), but also can achieve high penetration depth (up to 700-1000μm) of tissue in situ three-dimensional imaging with the help of two-photon microscopy. These advantages make it very suitable for in situ research on the microenvironment of the entire tumor tissue. For example, Christian et al. reported the use of fluorescence-encoded CODEX technology to study the microenvironment of colorectal cancer, achieved simultaneous detection of 57 proteins, and analyzed 9 cell neighborhoods and their interactions in its immune microenvironment. Recently, Carole et al. reported the use of single-cell sequencing and in situ imaging of 23 protein markers based on CODEX to achieve Tfr heterogeneity analysis, not only found the key marker CD38 that can characterize the difference in Tfr cell origin, but also explained the functional determination mechanism of Tfr cells in different structures of tonsil 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 the release of immunosuppression and clinical precision immunotherapy for malignant tumors.

[0004] However, the emission spectrum of fluorescent dyes is relatively wide, and there is a certain overlap between the emission spectra of different dyes, which will cause crosstalk of fluorescent signals and affect the accuracy of target coding. Therefore, it is crucial to provide a fluorescence imaging system with low signal crosstalk for fluorescence-encoded spatial proteomics technology. Utility Model Content

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

[0006] An imaging system, comprising:

[0007] An excitation light source module that emits at least two different wavelengths of excitation light beams to excite a sample to be measured to generate N different wavelengths of emission light;

[0008] An imaging module that includes a filter set and an image sensor;

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

[0010] The filter set includes K filters, and each filter allows a corresponding one or more wavelengths of the emission light to pass through and blocks the emission light of other wavelengths. The imaging system includes a beam splitting module, and the N different wavelengths of the emission light are split by the beam splitting module and then pass through the corresponding filters and enter the corresponding image sensors for imaging;

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

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

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

[0014] Optionally,

[0015] The beam transmission module includes a first dichroic mirror and a second dichroic mirror, and the first dichroic mirror reflects the excitation light beam to the second dichroic mirror;

[0016] The lens module has an optical axis, and 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,

[0017] The dichroic mirror allows the N different wavelengths of the emission light to pass through and blocks other light beams from passing through.

[0018] Optionally, the imaging system further includes a focusing module and a driving module;

[0019] The focusing module includes a focusing light source, a focusing sensor, a processor, and a driving module;

[0020] The focusing light source emits a focusing light beam, and the focusing light beam enters the lens module after passing through the first dichroic mirror and being reflected by the second dichroic mirror;

[0021] The focusing sensor receives the focusing light beam reflected from the sample to be measured;

[0022] The processor determines the defocus amount of the sample to be measured relative to the lens module based on the focusing light beam reflected from the sample to be measured received by the focusing sensor;

[0023] The driving module drives the lens module to move along its optical axis based on the defocus amount, so that the sample to be measured is located on the focal plane of the lens module and / or a clear image of the sample to be measured is obtained.

[0024] Optionally, 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.

[0025] Optionally, the filter set is located between the second dichroic mirror and the image sensor.

[0026] Optionally, a focusing lens is provided between the filter set and the image sensor.

[0027] In some embodiments, the excitation light source module includes N excitation light sources;

[0028] The N excitation light sources are sequentially turned on in a preset order, and each excitation light source excites the sample to be measured to generate one wavelength of emission light among N different wavelengths of emission light.

[0029] In some embodiments, the excitation light source module includes a first excitation light source and a second excitation light source;

[0030] The first excitation light source emits a first excitation light beam;

[0031] The second excitation light source emits a second excitation light beam;

[0032] The wavelength of the first excitation light beam is different from the wavelength of the second excitation light beam.

[0033] 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 at different times;

[0034] The first excitation light beam excites the sample to be measured to generate N - L different wavelengths of emission light among N different wavelengths of emission light; the second excitation light beam excites the sample to be measured to generate the remaining wavelengths of emission light;

[0035] Wherein, 1 ≤ L < N, and L is an integer.

[0036] In some embodiments, the imaging system includes a turntable, and M filters are arranged on the turntable. By rotating the turntable, the M filters enter the imaging optical path of the imaging system in a preset order.

[0037] In some embodiments, the first excitation beam and the second excitation beam sequentially excite the sample to be measured to generate five different wavelengths of emission light;

[0038] The first excitation beam excites the sample to be measured to generate emission light with a first wavelength, a second wavelength, and a third wavelength, and the second excitation beam excites the sample to be measured to generate emission light with a fourth wavelength and a fifth wavelength;

[0039] The image sensor includes a first image sensor, a second image sensor, and a third image sensor;

[0040] The beam splitting module includes:

[0041] A third dichroic mirror, which is opposite to the light-emitting surface of the second dichroic mirror, transmits the emission light with the first wavelength and the emission light with the fourth wavelength to the first image sensor, and reflects the remaining fluorescence;

[0042] A fourth dichroic mirror, which is opposite to the reflecting surface of the third dichroic mirror, reflects the emission light with the second wavelength and the emission light with the fifth wavelength to the second image sensor, and transmits the emission light with the third wavelength to the third image sensor.

[0043] In some embodiments, the filter set includes a first filter, a second filter, and a third filter;

[0044] The first filter is located between the third dichroic mirror and the first image sensor, and is opposite to the light-emitting surface of the third dichroic mirror, filters the emission light with the first wavelength and the emission light with the fourth wavelength, and the first image sensor is located on the light-emitting surface side of the first filter;

[0045] The second filter is located between the fourth dichroic mirror and the second image sensor, and is opposite to the reflecting surface of the fourth dichroic mirror, filters the emission light with the second wavelength and the emission light with the fifth wavelength, and the second image sensor is located on the light-emitting surface side of the second filter;

[0046] The third filter is located between the fourth dichroic mirror and the third image sensor. The third filter faces the light-transmitting surface of the fourth dichroic mirror and filters the emitted light of the third wavelength. The third image sensor is located on the light-emitting surface side of the third filter.

[0047] A sequencing system includes the imaging system according to any one of the above embodiments.

[0048] A spatial protein component analysis system includes the imaging system according to any one of the above embodiments.

[0049] Compared with the prior art, the beneficial effects of the technical solution of the present application are as follows:

[0050] The imaging system provided by the present application includes an excitation light source module and an imaging module. The imaging module includes a filter group and an image sensor. The filter group includes M filters, and each filter allows the emitted light of a corresponding wavelength to pass through and blocks the emitted light of other wavelengths. The M filters enter the imaging optical path of the imaging system in a preset order and transmit the emitted light of the corresponding wavelength, so that the emitted light of N different wavelengths enters the same image sensor for imaging in sequence; or the filter group includes K filters, and each filter allows the emitted light of a corresponding one or more wavelengths to pass through and blocks the emitted light of other wavelengths. The imaging system further includes a beam splitting module. The emitted light of N different wavelengths is split by the beam splitting module, then passes through the corresponding filters and enters the corresponding image sensors for imaging. It can be seen that the imaging system can either make the emitted light of different wavelengths enter the same image sensor in sequence through the filters, or make the emitted light of different wavelengths enter the corresponding different image sensors through the filters and the beam splitting module. Furthermore, the emitted light of each wavelength has a corresponding image sensor, that is, the emitted light of each wavelength is imaged separately, so that the signal crosstalk between the emitted lights of different wavelengths can be suppressed, the fluorescence signal crosstalk of the imaging system is relatively low, and the imaging quality of the sample to be measured can be improved. Description of the Drawings

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the related art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0052] The structures, proportions, sizes, etc. shown in the accompanying drawings of this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the conditions for the implementation of this application. Therefore, they do not have any substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the efficacy that this application can produce and the purpose that can be achieved, should still fall within the scope covered by the technical content disclosed in this application.

[0053] Figure 1 are fluorescence spectra of different emitted lights;

[0054] Figure 2 is a schematic structural diagram of an imaging system provided by this application;

[0055] Figure 3 is a schematic structural diagram of another imaging system provided by this application;

[0056] Figure 4 is a schematic structural diagram of yet another imaging system provided by this application;

[0057] Figure 5 is a schematic structural diagram of yet another imaging system provided by this application. Detailed implementation manners

[0058] Next, the embodiments in this application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope protected by this application.

[0059] To make the above objects, features, and advantages of this application more obvious and understandable, the following further details this application in conjunction with the drawings and specific implementation manners.

[0060] As described in the background art section, due to the relatively wide emission spectrum of fluorescent dyes, there will be an overlap in the emission spectra of different dyes. For example Figure 1 as shown, this will cause signal crosstalk between different fluorescences, resulting in mutual influence on the imaging of different fluorescences, poor imaging quality, and further affecting the accuracy of target coding.

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

[0062] An excitation light source module 100 emits at least two different wavelengths of excitation light beams to excite a sample to be measured to generate N different wavelengths of emission light, that is, the excitation light beams emitted by the excitation light source module 100 can laser the sample to be measured to generate emission light, thus generating N different wavelengths of emission light.

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

[0064] Based on the above, in an embodiment of the present application, as Figure 3 shown, the filter set 210 includes M filters 211. Each filter 211 allows the transmission of emission light of a corresponding one wavelength and blocks the emission light of other wavelengths. For example, the N different wavelengths of emission light are A, B, C, D, and E respectively. The first filter among the M filters 211 allows the emission light A to pass through and blocks the emission light B, C, D, and E from continuing to transmit through it. The second filter allows the emission light B to pass through and blocks the emission light A, C, D, and E from continuing to transmit through it, and so on. The M filters 211 enter the imaging optical path of the imaging system in a preset order and transmit the emission light of the corresponding wavelength, so that the N different wavelengths of emission light enter the same image sensor 220 in sequence. Among them, M = N, and M, N ≥ 2, and M and N are both integers.

[0065] As can be seen from the above, in this embodiment, when the imaging system works, the M filters 211 will enter the imaging optical path in sequence, and each filter 211 only allows the transmission of emission light of a corresponding one wavelength. Thus, as the M filters 211 enter the imaging optical path in sequence, the above N different wavelengths of emission light can enter the image sensor 220 in sequence to image the sample to be measured. Since as the M filters 211 enter the imaging optical path in sequence, the N different wavelengths of emission light will also enter the image sensor 220 in sequence, then the N different wavelengths of emission light will not enter the imaging optical path at the same time, and thus will not enter the same image sensor 220 at the same time, that is, the N different wavelengths of emission light will not image the sample to be measured at the same time. Therefore, theoretically, there is no signal crosstalk between the emission lights of different wavelengths, which 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 relatively low and has a better imaging effect, which is of great significance for the fluorescence-coded spatial proteomics technology.

[0066] In another embodiment of the present application, as Figure 2As shown, the filter group 210 includes K filters 211. Each filter 211 allows the emitted light of one or more corresponding wavelengths to pass through and blocks the emitted light of other wavelengths. Based on this, the imaging system further includes a beam splitting module 300. After the emitted light of N different wavelengths is split by the beam splitting module 300, it passes through the corresponding filter 211 and enters the corresponding image sensor 220 for imaging, that is, the emitted light of each wavelength enters a corresponding image sensor 220 for imaging. Among them, 2 ≤ K ≤ N, and both N and K are integers. Based on the description of this embodiment, the difference between this embodiment and the above embodiment is that: in this embodiment, there are image sensors 220 corresponding one by one to the emitted light of N different wavelengths. After the emitted light of N different wavelengths is filtered by the filter 211 and split by the beam splitting module 300 in sequence, it can enter the corresponding image sensors 220 for imaging respectively, so there is no crosstalk problem between the emitted lights of different wavelengths. Therefore, the imaging system can effectively suppress the signal crosstalk between the emitted lights of different wavelengths, so that the fluorescence signal crosstalk of the imaging system is relatively low.

[0067] In summary, the imaging system provided by the present application can either make the emitted light of different wavelengths enter the same image sensor in sequence through the filter, or make the emitted light of different wavelengths enter the corresponding different image sensors through the filter and the beam splitting module. Furthermore, each wavelength of the emitted light has a corresponding image sensor, that is to say, each wavelength of the emitted light is imaged separately, which can suppress the signal crosstalk between the emitted lights of different wavelengths, making the fluorescence signal crosstalk of the imaging system relatively low and improving the imaging quality of the sample to be measured.

[0068] On this basis, in an embodiment of the present application, the imaging system further includes a lens module 400. For example, the lens module 400 includes an objective lens, etc., and a beam transmission module 500 is provided between the excitation light source module 100 and the lens module 400, so that the excitation beam emitted by the excitation light source module 100 can be transmitted to the sample to be measured through the beam transmission module 500 to excite the sample to be measured to generate the emitted light of N different wavelengths.

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

[0070] Based on the above, when the imaging system works, the excitation light source module 100 emits an excitation beam. After the excitation 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. Thus, the beam transmission module 400 can transmit the excitation beam emitted by the excitation light source module 100 to the lens module 400, and then transmit it to the sample to be measured through the lens module 400, exciting the sample to be measured to generate N kinds of emission light with different wavelengths.

[0071] In addition, in this embodiment, the second dichroic mirror 520 also allows N kinds of emission light with different wavelengths to pass through and blocks other beams from passing through. That is to say, the second dichroic mirror 520 can allow N kinds of emission light with different wavelengths to pass through, but blocks the beams other than the emission light from continuing to transmit through it. Thus, after the excitation beam excites the sample to be measured to generate N kinds of emission light with different wavelengths, the second dichroic mirror 520 can block the beams other than the above N kinds of emission light with different wavelengths from entering the imaging optical path, which can suppress the signal crosstalk between the excitation beam and the emission beam and help improve the imaging quality of the imaging system.

[0072] Based on the above embodiments, in an embodiment of the present application, as Figure 4 shown, the imaging system further includes a focusing module 600 and a driving module 700. 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 enters the lens module 400 through the second dichroic mirror 520.

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

[0075] The processor 630 determines the defocus amount of the sample to be measured relative to the lens module 400 based on the focus beam reflected from the sample to be measured received by the focus sensor 620. This defocus amount is the distance between the plane where the sample to be measured is located, that is, the surface of the sample to be measured, 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 measured is located on the focal plane of the lens module 400 and / or a clear image of the sample to be measured is obtained. That is to say, the driving module 700 drives the lens module 400 to move along its optical axis so that the sample to be measured 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 measured, or the driving module 700 drives the lens module 400 to move along its optical axis so that the sample to be measured is located on the focal plane of the lens module 400 and at the same time a clear image of the sample to be measured is obtained. 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 measured is located on the focal plane of the lens module 400 and / or a clear image of the sample to be measured is obtained, in actual applications, it is also possible to drive the sample to be measured to move along the optical axis of the lens module 400 by the driving module 700 so that the sample to be measured is located on the focal plane of the lens module 400 and / or a clear image of the sample to be measured is obtained, which can be determined according to the specific situation.

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

[0078] Based on the above embodiment, in an embodiment of the present application, as Figure 5As shown, the imaging system further includes a collimating mirror. A collimating mirror is disposed between the focusing module 600 and the first dichroic mirror 510, and / or a collimating mirror is disposed between the excitation light source module 100 and the first dichroic mirror 510. For example, the collimating mirror may include a first collimating mirror 650 and / or a second collimating mirror 660. A first collimating mirror 620 is disposed between the focusing module 600 and the first dichroic mirror 510 to collimate the focusing beam and improve the directivity of the collimated beam. Or a second collimating mirror 660 is disposed between the excitation light source module 100 and the first dichroic mirror 510 to collimate the excitation beam and improve the directivity of the excitation beam. Or a first collimating mirror 650 is disposed between the focusing module 600 and the first dichroic mirror 510, and a second collimating mirror 660 is disposed 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 their directivities, which helps to ensure the imaging quality of the imaging system.

[0079] Based on the above embodiments, the filter set 210 is located between the second dichroic mirror 520 and the image sensor 220, so that the emitted 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, N different wavelengths of emitted light allowed by the second dichroic mirror 520 and transmitted through the second dichroic mirror 520 enter the same image sensor 220 through the corresponding filters 211 for imaging, or N different wavelengths of emitted light allowed by the second dichroic mirror 520 and transmitted through the second dichroic mirror 520 enter N image sensors respectively after passing through the filters 211 and the beam splitting module 300 for imaging.

[0080] Based on the above embodiments, in an embodiment of the present application, a focusing lens 700 is disposed between the filter set 210 and the image sensor 220 to converge the emitted light transmitted through the filters 211 in the filter set 210 to the image sensor 220 for imaging.

[0081] Based on the above embodiments, in an embodiment of the present application, for the case where N different wavelengths of emitted light correspond to the image sensor one by one, such as Figure 5As shown, 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 sequentially turned on in a preset order, and each of the N excitation light sources 110 in the N excitation light sources 110 excites the sample to be measured to generate one of the N emission lights with different wavelengths. That is to say, the N excitation light sources 110 correspond one-to-one with the N emission lights with different wavelengths. Therefore, during the operation of the imaging system, the N excitation light sources 110 can be sequentially turned on in a preset order, and thus can sequentially excite the sample to be measured one by one to generate the N emission lights with different wavelengths, 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 with different wavelengths and improving the imaging quality of the imaging system. It should be noted that since the emission lights with different wavelengths can be excited by excitation beams with the same or similar wavelengths, the wavelengths of the N excitation beams emitted by the above N excitation light sources can be different from each other, or some of the N excitation beams can have the same wavelength, depending on the wavelengths of the excitation beams corresponding to the N emission lights with different wavelengths.

[0082] In another embodiment of the present application, as Figure 5 shown, 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 used to emit a first excitation beam, and the second excitation light source 130 is used to emit a second excitation beam. Among them, the wavelength of the first excitation beam is different from the wavelength of the second excitation beam.

[0083] According to the description of this embodiment, the difference between this embodiment and the above embodiment is that the excitation light source module 100 in the above embodiment has excitation light sources corresponding one-to-one with N emission lights with different wavelengths, that is, the excitation light source module 100 in the above embodiment has N excitation light sources 110. However, in practical applications, the emission lights with different wavelengths can be excited by excitation beams with the same wavelength. Therefore, in this embodiment, the excitation light source module 100 can include two excitation light sources, namely the first excitation light source 120 and the second excitation light source 130. That is to say, the excitation light source module 100 can include fewer excitation light sources than the number of emission lights, so that the number of excitation light sources in the excitation light source module 100 can be reduced, and thus the volume of the excitation light source module 100 can be reduced while reducing the cost of the excitation light source module 100, and the volume of the imaging system is also reduced and the cost is lowered. 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 the actual situation, 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] Based on the above embodiments, in an embodiment of the present application, the first excitation light source 120 and the first excitation light source 130 emit the first excitation beam and the second excitation beam at different times. That is to say, the first excitation beam and the second excitation beam are not emitted simultaneously, but are emitted in sequence. For example, the first excitation light source 120 emits the first excitation beam first, and then the second excitation light source 130 emits the second excitation beam, or the second excitation light source 130 emits the second excitation beam first, and then the first excitation light source 120 emits the first excitation beam.

[0085] And the first excitation beam excites the sample to be measured to generate N - L different wavelengths of the N different wavelengths of the emitted light, and the second excitation beam excites the sample to be measured to generate the remaining wavelengths of the emitted light, where 1 ≤ L < N, and L is an integer. That is to say, the first excitation beam emitted by the first excitation light source 120 can excite the sample to be measured to generate the emitted light of some wavelengths, and the second excitation beam emitted by the second excitation light source 130 can excite the sample to be measured to generate the emitted light of the remaining wavelengths. Thus, the first excitation light source 120 and the first excitation light source 130 emit the first excitation beam and the second excitation beam at different times, and can sequentially excite the sample to be measured to generate N different wavelengths of the emitted light for imaging the sample to be measured, suppressing the mutual interference between the emitted lights of different wavelengths, and improving the imaging quality of the sample to be measured.

[0086] Based on the filter set 210 including M filters 211, in an embodiment of the present application, the imaging system includes a turntable 2111, and the M filters 211 are arranged on the turntable 2111. By rotating the turntable 2111, the M filters can enter the imaging optical path of the imaging system in sequence, allowing the emitted light of the corresponding wavelengths among the N different wavelengths of the emitted light to pass through, while blocking the emitted light of the remaining wavelengths, thereby realizing the isolation of the N different wavelengths of the emitted light, suppressing the interference between the N different wavelengths of the emitted light, and ensuring the imaging quality of the sample to be measured.

[0087] Taking the example that the excitation beam excites the sample to be measured to generate five different wavelengths of the emitted light, which are respectively denoted as the emitted light of the first wavelength, the emitted light of the second wavelength, the emitted light of the third wavelength, the emitted light of the fourth wavelength, and the emitted light of the fifth wavelength. Based on the filter set 210 including K filters 211, in an embodiment of the present application, the first excitation beam and the second excitation beam sequentially excite the sample to be measured, causing the sample to be measured to generate five different wavelengths of the emitted light. For example, the first excitation beam can excite the sample to be measured to generate the emitted light of the first wavelength, the emitted light of the second wavelength, and the emitted light of the third wavelength, and the second excitation beam excites the sample to be measured to generate the emitted light of the fourth wavelength and the emitted light of the 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 beam splitting module 300 includes a third dichroic mirror 310 and a fourth dichroic mirror 320.

[0089] The third dichroic mirror 310 faces the light-emitting surface of the second dichroic mirror 520, transmits the emitted light of the first wavelength and the emitted light of the fourth wavelength to the first image sensor 211, and blocks the emitted light of the remaining wavelengths.

[0090] The fourth dichroic mirror 320 faces the reflecting surface of the third dichroic mirror 310, reflects the emitted light of the second wavelength and the emitted light of the fifth wavelength to the second image sensor 222, and the fourth dichroic mirror 320 also transmits the emitted light of the third wavelength to the third image sensor 223.

[0091] Based on the above, the first excitation light source 120 emits a first excitation beam to excite the sample to be measured to generate the emitted light of the first wavelength, the emitted light of the second excitation wavelength, and the emitted light of the third excitation wavelength. Then, the emitted light of these three wavelengths is transmitted by the second dichroic mirror 520 to the third dichroic mirror 310. Since the third dichroic mirror 310 only transmits the emitted light of the first wavelength and the emitted light of the fourth wavelength, when the emitted light of these three wavelengths is transmitted to the third dichroic mirror 310, only the emitted light of the first wavelength is transmitted to the first image sensor for imaging, and the emitted light of the second wavelength and the emitted light of the third wavelength will be reflected to the fourth dichroic mirror 320. Since the fourth dichroic mirror 320 only transmits the emitted light of the third wavelength, the fourth dichroic mirror 320 can reflect the emitted light of the second wavelength transmitted to it to the second image sensor 222 for imaging, and transmit the emitted light of the third wavelength to the third image sensor 223 for imaging, so that the emitted light of the first wavelength, the emitted light of the second wavelength, and the emitted light of the third wavelength that are simultaneously excited enter different image sensors for imaging, thus avoiding signal crosstalk between the emitted lights of different wavelengths and improving the imaging quality of the sample to be measured.

[0092] Similarly, the second excitation light source 130 emits a second excitation beam to excite the sample to be measured to generate the emitted light of the fourth wavelength and the emitted light of the fifth wavelength. When the emitted light of the fourth wavelength and the emitted light of the fifth wavelength are transmitted through the lens module 400 to the third dichroic mirror 310, the emitted light of the fourth wavelength will be transmitted by the third dichroic mirror 310 to the first image sensor for imaging, and the emitted light of the fifth wavelength will be reflected by the third dichroic mirror 320 to the fourth dichroic mirror 320 and then reflected by the fourth dichroic mirror 320 to the second image sensor 222 for imaging, which also makes the emitted light of the fourth wavelength and the emitted light of the fifth wavelength generated simultaneously enter different image sensors, suppressing the interference between the emitted lights of different wavelengths and improving the imaging quality of the sample to be measured.

[0093] Based on the above embodiments, in an embodiment of the present application, as Figure 5 shown, the filter group 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 faces the light-emitting surface of the third dichroic mirror 310, and is used for filtering the emitted light of the first wavelength and the emitted light of the fourth wavelength. Moreover, the first image sensor 221 is located on the light-emitting surface side of the first filter 212. Therefore, the emitted light of the first wavelength and the emitted light of the fourth wavelength transmitted through the third dichroic mirror 310 will enter the first image sensor 221 after being filtered by the first filter, effectively suppressing the entry of light beams other than the emitted light of the first wavelength and the emitted light of the fourth wavelength into the first image sensor 221, reducing the imaging influence of the remaining light beams on the emitted light of the first wavelength and the emitted light of the fourth wavelength, and 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, and the second filter 213 faces the reflective surface of the fourth dichroic mirror 320, and is used for filtering the emitted light of the second wavelength and the emitted light of the fifth wavelength. The second image sensor 222 is located on the light-emitting surface side of the second filter 213. Since the second filter 213 faces the reflective surface of the fourth dichroic mirror 320, the emitted light of the second wavelength and the emitted light of the fifth wavelength reflected by the fourth dichroic mirror 320 will be transmitted to the second filter 213, and after being filtered by the second filter 213, will enter the second image sensor 222. Similarly, it can effectively suppress the entry of light beams other than the emitted light of the second wavelength and the emitted light of the fifth wavelength into the second image sensor 222, reducing the imaging influence of the remaining light beams on the emitted light of the second wavelength and the emitted light of the fifth wavelength, and improving the imaging quality of the sample to be measured.

[0095] The third filter 214 is located between the fourth dichroic mirror 320 and the third image sensor 223, and the third filter 214 faces the light-transmitting surface of the fourth dichroic mirror 320, and is used for filtering the emitted light of the third wavelength. Moreover, the third image sensor 223 is located on the light-emitting surface side of the third filter 214. Therefore, the emitted light of the third wavelength transmitted through the fourth dichroic mirror 320 will be filtered by the third filter 214 and then enter the third image sensor 223, reducing the imaging influence of the remaining light beams on the emitted light of the third wavelength, and 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, and the wavelengths of the excitation light beams are 488 nm, 532 nm, and 660 nm respectively. The N kinds of emission lights with different wavelengths are five kinds of emission lights generated by exciting five fluorescent dyes, namely Sytox Green, ATTO532, ROX, CY5, and IF700, with the above three excitation light beams, and the wavelengths are 512 nm, 551 nm, 602 nm, 670 nm, and 712 nm respectively. The scanning range of the imaging system can be 20 mm × 20 mm, the imaging mode can be wide-field fluorescence imaging, the spatial resolution is ≤ 600 nm, the magnification can be 12.8, the image size can be 4112 pixels × 2176 pixels, the object space field of view can be 1.10 mm × 0.58 mm, the imaging speed is ≤ 400 ms / field of view, the stage field of view switching time is ≤ 80 ms / field of view, and the stage positioning accuracy is ≤ 200 nm. Among them, the sample to be tested is placed on the stage, and the sample to be tested can be a paraffin-embedded or frozen tissue section or a nucleic acid sample, etc.

[0097] Based on the imaging system described in any of the above embodiments, the present application also provides a sequencing system, and the sequencing system includes the imaging system described in any of the above embodiments. The sequencing system includes the imaging system, aiming to suppress the crosstalk problem between the emission lights of different wavelengths when the sequencing system works, ensure the imaging quality of the images used for sequencing analysis, and thus contribute to 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, and the spatial protein analysis system includes the imaging system described in any of the above embodiments. Since the imaging system can suppress the crosstalk problem between the emission lights of different wavelengths, that is, it can suppress the fluorescence signal crosstalk problem during spatial protein analysis, which helps to ensure the accuracy of spatial protein analysis.

[0099] It should be noted that the above sequencing system and spatial protein analysis system have improved the structure of their imaging systems and suppressed the fluorescence signal crosstalk problem during imaging. However, the rest of the working processes of the sequencing system and spatial protein analysis system, such as sequencing, etc., will not change. Therefore, the specific working process of the sequencing system in the present application will not be described in detail.

[0100] In summary, the present application provides an imaging system, a sequencing system, and a spatial protein analysis system. The imaging system includes an excitation light source module and an imaging module. The imaging module includes 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 optical path of the imaging system in a preset order and transmit the emission light of the corresponding wavelength, so that the emission light of N different wavelengths enters the same image sensor for imaging in sequence. Or the filter set includes K filters, each of which allows the emission light of a corresponding one or more wavelengths to pass through and blocks the emission light of other wavelengths. The imaging system further includes a beam splitting module. The emission light of N different wavelengths is split by the beam splitting module, then passes through the corresponding filters and enters the corresponding image sensors for imaging. Thus, it can be seen that the imaging system can either make the emission light of different wavelengths enter the same image sensor in sequence through the filters, or make the emission light of different wavelengths enter the corresponding different image sensors through the filters and the beam splitting module. Furthermore, each wavelength of emission light has a corresponding image sensor, that is, each wavelength of emission light is imaged separately, so that the signal crosstalk between the emission lights of different wavelengths can be suppressed, the fluorescence signal crosstalk of the imaging system is relatively low, and the imaging quality of the sample to be measured can be improved.

[0101] In the present specification, the various embodiments are described in a progressive, or parallel, or a combination of progressive and parallel manners. The key point of each embodiment is to illustrate the differences from other embodiments. For the same or similar parts among the embodiments, reference can be made to each other. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple. For the relevant parts, reference can be made to the description in the method section.

[0102] It should be noted that in the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present 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 intermediate components present simultaneously.

[0103] It should also be noted that, in this text, relational terms such as first and second are only used 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 term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that an article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the article or device comprising the above elements.

[0104] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

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

2. The imaging system according to claim 1, characterized in that The imaging system also includes a lens module; A light beam transmission module is arranged between the excitation light source module and the lens module.

3. The imaging system according to claim 2, characterized in that The beam transmission module comprises 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, characterized in that The imaging system also 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 light beam, and the focusing light beam passes through the first dichroic mirror and then is reflected by the second dichroic mirror and enters the lens module; The focus sensor receives a focus beam reflected from the sample to be tested; The processor determines the defocus amount of the sample to be tested relative to the lens module based on the focus light beam reflected from the sample to be tested and received by the focus sensor; 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, characterized in that: A collimator is arranged 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 claim 3, characterized in that: The filter group is located between the second dichroic mirror and the image sensor.

7. The imaging system according to claim 6, characterized in that: 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, characterized in that: 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 source 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, characterized in that The first excitation light source and the second excitation light source emit the first excitation light beam and the second excitation light beam in different time periods; The first excitation light beam excites the sample to be tested to generate NL types of emission lights with different wavelengths among N types of emission lights with 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 10, characterized in that: The imaging system comprises a turntable, and the M optical filters are arranged on the turntable. When the turntable is rotated, the M optical filters enter the imaging optical path of the imaging system in a preset order.

12. The imaging system according to claim 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 the emission light of the first wavelength, the emission light of the second wavelength, and the emission light of the third wavelength, and the second excitation light beam excites the sample to be tested to generate the emission light of the fourth wavelength and the emission light of the fifth wavelength; The image sensor includes a first image sensor, a second image sensor and a third image sensor; The optical splitting module comprises: a third dichroic mirror, the third dichroic mirror being opposite to the light exiting 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 reflection surface of the third dichroic mirror, reflects the emission light of the second wavelength and the emission light of the fifth wavelength to the second image sensor, and transmits the emission light of the third wavelength to the third image sensor.

13. The imaging system according to claim 12, characterized in that: 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 filters the emission light of the first wavelength and the emission light of the fourth wavelength, and 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 filters 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 filters the emission light of the third wavelength. The third image sensor is located on the light-emitting surface side of the third filter.

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

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

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