Detection device, gene sequencing system, and detection method

Abstract

The present disclosure provides a detection device, a system, and a method, which belong to the technical field of detection. The detection device includes a beam splitting device, a first dichroic mirror, an objective lens, a fluorescence induction device, and an imaging system including a plurality of imaging devices. The beam splitting device receives and splits an incident light beam of a plurality of different wavelengths formed by a laser from an optical fiber, and forms a plurality of excitation beams corresponding to the plurality of different wavelengths of the incident light beam and emitted from the beam splitting device in different emission directions. The first dichroic mirror receives the plurality of excitation lasers emitted from the beam splitting device and transmits them to the objective lens, receives a plurality of fluorescences and transmits them to the fluorescence induction device, the objective lens receives the plurality of excitation lasers and focuses them on the plurality of different regions of the sample respectively to excite fluorescence, and transmits the plurality of fluorescences to the first dichroic mirror, and the fluorescence induction device receives the plurality of fluorescences transmitted through the first dichroic mirror and induces the plurality of fluorescences to the plurality of imaging devices respectively. The present disclosure can reduce the power density of the laser on the sample without increasing the detection duration, solving the problem that the two aspects of "high speed" and "high detection quality and long read length" cannot be well combined in related technologies.

Description

[Technical Field]

[0001] This disclosure relates to the technical field of detection, and more particularly to detection devices, gene sequencing systems, and detection methods. [Background technology]

[0002] In the field of biochemical detection, it is known that a sample can be excited by irradiating it with a laser to generate fluorescence, and the desired detection of the sample can be achieved by detecting the generated fluorescence. Below, we will introduce some related techniques, using gene detection as an example.

[0003] Gene detection is completed by exciting a sample with a laser to generate fluorescence, and then detecting and analyzing the generated fluorescence using an imaging system to obtain the target base sequence. Various bases present on the sample to be detected are labeled with different primers, traced using primers excited with lasers of different wavelengths, and the corresponding fluorescence (one-to-one correspondence with the base) is obtained. During the fluorescence excitation process, there are associated phenomena such as photobleaching and fluorescence crosstalk. Photobleaching significantly reduces the fluorescence yield and even denatures the fluorescence, degrading the detection results. Fluorescence crosstalk refers to the phenomenon where, when excited to obtain excitation fluorescence, some primers are affected by the excitation process of other primers, influencing the quantum yield, resulting in the simultaneous generation of multiple fluoresces in one region and mixing with each other. The higher the power density of the excitation light, the longer the excitation duration, and the longer the detection reading length, the more pronounced the phenomena of photobleaching and fluorescence crosstalk become.

[0004] In pursuit of high speed, high throughput, and low cost, the laser power density used in detection systems is increasing, and accompanying phenomena such as photobleaching and fluorescence crosstalk seriously affect sequencing quality, limiting the detectable base sequence length (read length) by gene detection. In particular, new generation high-speed and high-throughput gene detection technologies employ TDI (Time Delay Integration) imaging technology, which uses a method of high-speed scanning by simultaneously exciting fluorescence with multiple wavelength lasers, and the laser power density used is hundreds of times higher than that of conventional area array imaging methods. As the detection read length increases, the effects of high-power-density lasers become increasingly pronounced, leading to a rapid deterioration of detection quality. Related gene detection technologies cannot successfully achieve both "high speed" and "high detection quality and long read length."

[0005] Conventional gene detection devices output lasers of different wavelengths coaxially through optical fibers to reduce illumination costs and structural complexity. As the beams pass through the beam shaping device and objective lens, the principal rays of all wavelengths are transmitted coaxially and ultimately irradiate the same area of ​​the sample. Two options are available: simultaneous excitation and time-resolved excitation.

[0006] Figure 6 shows a known apparatus 60 for implementing the first related technique (simultaneous excitation). As shown in Figure 6, when the first related technique (simultaneous excitation) is employed, the multimode optical fiber 610 simultaneously outputs n lasers of different wavelengths (n is the number of lasers of different wavelengths, the same applies hereafter). These lasers are shaped by a beam shaping device 670, filtered by a laser filter 620, reflected by a first dichroic mirror 630, focused by an objective lens 640, and then focused onto a sample 600. Subsequently, the n lasers of different wavelengths are focused onto the same location on the sample (the location where the spot formed by the multiple wavelength lasers in Figure 6 exists), exciting fluorescence at that location on the sample. The fluorescence is detected by the objective lens 640, passes through the first dichroic mirror 630, then through other specific dichroic mirrors constituting the fluorescence induction device 650 (dichroic mirror 1, dichroic mirror 2, ..., dichroic mirror n), and then through specific optical filters (optical filter 1, optical filter 2, ..., optical filter n, each optical filter transmitting only fluorescence of a specific wavelength), where fluorescence of different wavelengths is spectrally separated. This fluorescence of different wavelengths then passes through imaging lenses corresponding to the different wavelengths of fluorescence in the imaging system 660 (imaging lens 1, imaging lens 2, ..., imaging lens n), and is captured by cameras corresponding to the imaging lenses in the imaging system 660 (camera 1, camera 2, ..., camera n) to output fluorescence information. Fluorescence excited by the nth laser passes through the corresponding nth optical filter, is focused by imaging lens n, and then received by camera n. In this way, the process of simultaneous excitation and detection of fluorescence can be completed. The fluorescence detection rate in each field of view (FOV) is equal to the time of a single shot of the camera, and the laser output in the illumination region is the sum of the outputs of all lasers; that is, the density is n times greater than that of illumination by a single laser (assuming the laser power density is the same for each wavelength). At high power densities, fluorescence is more severely affected by photobleaching, and fluorescence crosstalk exists between multiple different channels, both of which affect the accuracy of the detection results.In conclusion, the first related technology focuses on "high speed," in which fluorescence is simultaneously excited by n lasers of different wavelengths, and fluorescence detection for all channels can be completed in a single shot in a single FOV region, but the power density is n times that of irradiation with a single wavelength laser.

[0007] Figure 7 shows a known apparatus 70 for implementing the second related technique (time-resolved excitation). As shown in Figure 7, when the second related technique (time-resolved excitation) is employed, the multimode optical fiber 710 sequentially outputs n lasers of different wavelengths. The output lasers are shaped by the beam shaping device 770, filtered by the laser filter 720, reflected by the dichroic mirror 730, focused by the objective lens 740, etc., and then focused to the position of the sample 700 (the position where the laser spots at different times in Figure 7 exist), exciting fluorescence at that position on the sample. The fluorescence is detected by the objective lens 740, passes through the dichroic mirror 730, the fluorescence filter 750 (which transmits only fluorescence and removes stray light other than fluorescence), and the imaging lens 760, and then captured by the camera 780 to output fluorescence information. Fluorescence detection of the FOV can be completed in only n shots. All lasers must be output sequentially by the multimode optical fiber, and fluorescence detection is performed sequentially. In other words, the power density of the lasers irradiated onto the sample is the same as that of irradiation with a single laser (assuming the power density of each laser is the same). Therefore, the phenomenon of photobleaching is relatively weak, and crosstalk between fluoresces is reduced. However, the fluorescence detection time for each FOV increases to n times the fluorescence detection time for a single shot. In conclusion, the second related technique focuses on "high detection quality and long read length," and its method involves sequentially exciting fluorescence with n lasers of different wavelengths (simultaneously, corresponding cameras take images for fluorescence detection in a one-to-one correspondence), with the same power density as when a single laser excites fluorescence, but requiring n excitations and n fluorescence detections, the total duration of fluorescence detection is n times that of the first related technique, and the total detection time required to detect the same sample is n times that of the first related technique.

[0008] The first related technology improves detection speed, but the laser irradiation power density increases by n times, causing different fluorescence to be excited at the same location, resulting in significant photobleaching, fluorescence crosstalk, and a decrease in fluorescence yield. As the readout length increases, the second related technology is clearly superior to the first related technology in terms of detection error rate, but the shot time is n times longer than that of the first related technology.

[0009] Therefore, an improved solution for biochemical detection is needed to address or mitigate the problems of photobleaching and fluorescence crosstalk caused by high optical power density, while also taking detection speed into full consideration. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The purpose of this disclosure is to provide an improved solution for biochemical detection that solves or mitigates the problems of photobleaching and fluorescence crosstalk caused by high optical power density, while giving due consideration to the detection rate. [Means for solving the problem]

[0011] According to a first aspect of this disclosure, the detection device includes a beam splitting device, a first dichroic mirror, an objective lens, a fluorescence induction device, and an imaging system including a plurality of imaging devices.

[0012] The beam splitting device receives and splits incident light rays of multiple different wavelengths from an optical fiber, forming multiple excitation rays that are emitted from the beam splitting device in different exit directions corresponding to the multiple different wavelengths of the incident light rays, and each of the excitation rays is configured to have a wavelength that corresponds one-to-one with the multiple different wavelengths of the incident light rays.

[0013] The first dichroic mirror is configured to receive the plurality of excitation rays emitted from the beam splitting device, which correspond one-to-one with the plurality of different wavelengths of the incident light, transmit the plurality of excitation rays to the objective lens, and focus the plurality of excitation rays through the objective lens onto a plurality of different regions of the sample to be detected, thereby exciting a plurality of fluorescence molecules in the plurality of different regions of the sample where the plurality of excitation rays are focused, and to receive the plurality of fluorescence molecules excited by the plurality of excitation rays and transmit the plurality of fluorescence molecules to the fluorescence induction device.

[0014] The objective lens is arranged to receive the plurality of excitation rays transmitted by the first dichroic mirror, to focus the plurality of excitation rays onto the plurality of different regions of the sample, and to transmit the plurality of fluorescence excited by the plurality of excitation rays to the first dichroic mirror.

[0015] The fluorescence induction device is configured to receive the plurality of fluorescence transmitted by the first dichroic mirror, to induce the plurality of fluorescence to each of the plurality of imaging devices, and to image each of the plurality of fluorescence by one of the plurality of imaging devices corresponding to the fluorescence.

[0016] Each of the plurality of imaging devices is arranged to receive one of the plurality of fluorescence induced by the fluorescence induction device, image the fluorescence, and obtain fluorescence information corresponding to the fluorescence to be detected.

[0017] According to a second aspect of this disclosure, a gene sequencing system includes an imaging system for collecting fluorescent signals on a sequencing chip and an optical system disposed between the imaging system and the sequencing chip, the optical system including:

[0018] A light source configured to emit incident light rays of multiple different wavelengths.

[0019] A beam splitting device configured to receive and split incident light rays of a plurality of different wavelengths from the light source, and to form a plurality of excitation light rays that are emitted in different emission directions from the beam splitting device corresponding to the plurality of different wavelengths of the incident light rays, wherein each of the excitation light rays has a wavelength corresponding one-to-one to the plurality of different wavelengths of the incident light rays.

[0020] A first dichroic mirror configured to receive the plurality of excitation light rays emitted from the beam splitting device corresponding one-to-one to the plurality of different wavelengths of the incident light rays.

[0021] An objective lens disposed between the array determination chip and the first dichroic mirror, configured to receive the plurality of excitation light rays transmitted by the first dichroic mirror, to condense the plurality of excitation light rays on a plurality of different regions on the array determination chip, to excite a plurality of fluorescences corresponding one-to-one to the plurality of excitation light rays in the plurality of different regions, and to transmit the plurality of fluorescences to the first dichroic mirror.

[0022] A fluorescence guiding device configured to receive the plurality of fluorescences transmitted through the first dichroic mirror and to guide the plurality of fluorescences to the imaging system respectively.

[0023] The imaging system includes a plurality of imaging devices corresponding one-to-one to the plurality of fluorescences. Each of the plurality of imaging devices is configured to receive one fluorescence corresponding to the imaging device among the plurality of fluorescences guided by the fluorescence guiding device, and to image the fluorescence to obtain fluorescence information corresponding to the fluorescence.

[0024] According to a third aspect of the present disclosure, the detection method includes the following steps.

[0025] A step of emitting incident light rays of a plurality of different wavelengths by a light source or an optical conduction device.

[0026] A beam splitting device receives and splits incident light rays of multiple different wavelengths from a light source or a photoconducting device, and forms a plurality of excitation rays that are emitted from the beam splitting device in different emission directions corresponding to the multiple different wavelengths of the incident light rays, wherein each of the excitation rays has a wavelength that corresponds one-to-one with the multiple different wavelengths of the incident light rays.

[0027] A step of receiving the plurality of excitation rays emitted from the beam splitting device, which correspond one-to-one with the plurality of different wavelengths of the incident light, using a first dichroic mirror.

[0028] The process involves an objective lens positioned between the sample to be detected and the first dichroic mirror to receive the plurality of excitation rays transmitted by the first dichroic mirror, focusing the plurality of excitation rays onto a plurality of different regions on the sample using the objective lens to excite a plurality of fluorescence molecules in each of the plurality of different regions in a one-to-one correspondence with the plurality of excitation rays, and transmitting the plurality of fluorescence molecules to the first dichroic mirror using the objective lens.

[0029] A step of receiving the plurality of fluorescence transmitted by the first dichroic mirror using a fluorescence induction device, inducing the plurality of fluorescence to a plurality of imaging devices that correspond one-to-one with the plurality of fluorescence using the fluorescence induction device, receiving and imaging each of the plurality of fluorescence with one of the plurality of imaging devices corresponding to the fluorescence, and obtaining fluorescence information corresponding to the fluorescence to be detected. [Effects of the Invention]

[0030] The configuration of the present invention enables simultaneous and separate irradiation of different wavelengths of light on different locations on the sample to be detected, thereby simultaneously exciting fluorescence at those different locations. Taking a laser as the excitation light as an example, the laser power density on the sample is reduced to 1 / n of the laser density in the case of simultaneous excitation, and fluorescence detection of all channels can be completed by only a single excitation in a single field of view. If the fluorescence is excited multiple times in the same area for detection (i.e., in the case of time-resolved excitation), the total detection time is equal to the time required for a single fluorescence excitation and detection. By using the solution of the present disclosure, the laser power density on the sample can be reduced without increasing the detection duration, solving the problem of the inability to successfully reconcile the two aspects of "high speed" and "high detection quality and long read length" in related technologies. That is, the problems of photobleaching and fluorescence crosstalk caused by high optical power density can be solved or mitigated while giving due consideration to the detection speed. [Brief explanation of the drawing]

[0031] [Figure 1a] Figure 1a is a schematic diagram of the configuration and structure of a detection device according to an exemplary embodiment of the present disclosure. [Figure 1b] Figure 1b is a schematic diagram of the configuration and structure of an exemplary fluorescence induction device that can be used in the detection device of the present disclosure. [Figure 1c] Figure 1c is a schematic diagram of the configuration and structure of another exemplary fluorescence induction device that can be used in the detection device of the present disclosure. [Figure 2] Figure 2 is a schematic diagram of the configuration and structure of a detection device according to another exemplary embodiment of the present disclosure. [Figure 3] Figure 3 is a schematic diagram showing a detection device according to yet another exemplary embodiment of the present disclosure. [Figure 4] Figure 4 is a schematic diagram illustrating the spots formed on the surface of a sample by two excitation lasers when detection is performed using the detection apparatus according to an exemplary embodiment of this disclosure. [Figure 5]Figure 5 is a schematic diagram illustrating the process of performing fluorescence integration scanning using TDI imaging technology in an exemplary embodiment. [Figure 6] Figure 6 schematically shows a known apparatus that employs related detection techniques that use simultaneous excitation. [Figure 7] Figure 7 schematically shows a known apparatus that employs related detection techniques using time-resolved excitation. [Figure 8a] Figure 8a shows the relationship between data quality Q30 and read length of gene detection results obtained by performing double-end testing on DNA base sequences under the same conditions using related techniques employing simultaneous excitation, related techniques employing time-resolved excitation, and the present disclosure. [Figure 8b] Figure 8b shows the relationship between the detection error rate and the read length of gene detection results obtained by performing double-end testing on DNA base sequences under the same conditions using related techniques employing simultaneous excitation, related techniques employing time-resolved excitation, and the present disclosure. [Modes for carrying out the invention]

[0032] Exemplary embodiments of this disclosure will be described in detail with reference to the accompanying drawings, which form part of this disclosure and serve to illustrate the principles of this disclosure together with the embodiments of this disclosure. For clarity and simplification, specific descriptions of known functional and structural details of the devices described herein will be omitted where they would obscure the subject matter of this disclosure.

[0033] As shown in Figure 1, the detection device 10 according to an exemplary embodiment of the present disclosure includes a beam splitting device 120, a first dichroic mirror 130, an objective lens 140, a fluorescence induction device 150, and an imaging system 160 including imaging devices 1 through i, where i is an integer greater than 1, representing the number of different wavelengths of the incident laser from the optical fiber.

[0034] The beam splitting device 120 is configured to receive and split multiple incident lasers of different wavelengths from the optical fiber, and to form corresponding excitation lasers (such as laser 1, laser 2, and laser i shown in Figure 1) when the multiple incident lasers of different wavelengths are emitted from the beam splitting device in different emission directions. The wavelength of each excitation laser is the same as the wavelength of the incident laser corresponding to the excitation laser. The beam splitting device may be implemented by various optical devices having dispersion capabilities or combinations of such optical devices. For example, the beam splitting device may be a single dispersion prism, multiple dispersion prisms, one or more diffraction gratings, etc., depending on the specific circumstances of the actual application and corresponding requirements, as will be described later. The optical fiber may be a single optical fiber, but is not limited to, for example, a coupled optical fiber. Although not essential, it is advantageous if the multiple incident lasers of different wavelengths are incident on the beam splitting device in the same incident direction. In Figure 1, the incident lasers are transmitted to the beam splitting device by the optical fiber, but it is also possible to provide the incident lasers to the beam splitting device using other optical transmission devices instead of the optical fiber. The optical fiber and other optical transmission devices may be built into the detection device or attached externally.

[0035] The first dichroic mirror 130 is configured to receive the multiple excitation lasers emitted from the beam splitting device, each corresponding to an incident laser of multiple different wavelengths, and to focus the multiple excitation lasers through the objective lens onto multiple different regions of the sample to be detected 100 (such as the locations of spot 1, spot 2, and spot i in Figure 1), transmitting the multiple excitation lasers to the objective lens so as to excite fluorescence in the corresponding regions of the sample where the multiple excitation lasers are focused. The first dichroic mirror 130 is configured to receive the multiple fluorescence excited by the multiple excitation lasers and transmit the multiple fluorescence to the fluorescence induction device. In Figure 1, the focused spots of laser 1, laser 2, and laser i on the sample are schematically shown as spot 1, spot 2, and spot i. Here, the sample to be detected may be any substance that can emit fluorescence when irradiated with a laser, and is not particularly limited, but examples include biological samples and chemical samples. The first dichroic mirror may be appropriately oriented so that it receives all of the multiple excitation lasers emitted from the beam splitting device, each corresponding to an incident laser of a plurality of different wavelengths, reflects them, and then focuses them all onto the plurality of different regions of the sample by the objective lens, and transmits all of the plurality of fluorescence excited by the plurality of excitation lasers and received from the sample via the objective lens to the fluorescence induction device via the first dichroic mirror. Under this premise, the orientation of the first dichroic mirror may be determined, for example, based on the emission angle of each excitation laser with respect to the beam splitting device and the objective lens. Depending on the situation, the first dichroic mirror may be positioned at different angles.

[0036] The objective lens 140 is arranged to receive the plurality of excitation lasers transmitted via the first dichroic mirror, focus each of the plurality of excitation lasers onto the plurality of different regions of the sample, and transmit the plurality of fluorescence excited by the plurality of excitation lasers to the first dichroic mirror. Generally, the objective lens is a lens group consisting of a single lens or a combination of multiple lenses. The lenses are not particularly limited, but examples include convex lenses, concave lenses, bonded lenses, etc.

[0037] The fluorescence induction device 150 is configured to receive the plurality of fluorescence transmitted via the first dichroic mirror, induce the plurality of fluorescence to the plurality of imaging devices, and image each of the plurality of fluorescence by one of the plurality of imaging devices corresponding to that fluorescence. The fluorescence induction device may be implemented in various ways. For example, the fluorescence induction device may be a plurality of second dichroic mirrors provided as needed, and the number of the plurality of second dichroic mirrors may be equal to the number of excitation lasers, i.e., i.e., i.e., i.e., depending on the situation, each second dichroic mirror may be configured to perform either one of the following: transmit a specific fluorescence and reflect the remaining fluorescence, or reflect a specific fluorescence and transmit the remaining fluorescence, so that the combination of the plurality of second dichroic mirrors achieves the desired induction of fluorescence as described above. Each of the plurality of fluorescence induced by the fluorescence induction device corresponds one-to-one with each of the plurality of imaging devices. According to one possible embodiment, the fluorescence induction device includes a plurality of second dichroic mirrors arranged in a series, the plurality of second dichroic mirrors including a final second dichroic mirror spaced apart from the first dichroic mirror, and at least one preceding second dichroic mirror positioned between the final second dichroic mirror and the first dichroic mirror, wherein the plurality of fluorescence received by the first dichroic mirror is sequentially propagated through the plurality of second dichroic mirrors, each of the preceding second dichroic mirrors being arranged to guide one of the plurality of fluorescence, of which at least one fluorescence incident on it, to a corresponding imaging device, and the remaining fluorescence of the at least one fluorescence to an adjacent next second dichroic mirror, the final second dichroic mirror being arranged to guide the fluorescence incident on it to a corresponding imaging device.

[0038] Specifically, referring to an example of a fluorescence induction device 150' shown in Figure 1b, the fluorescence induction device 150' includes a plurality of second dichroic mirrors arranged in the order of dichroic mirror 1, dichroic mirror 2, ..., dichroic mirror i. Of the plurality of second dichroic mirrors, the second dichroic mirror located closest to the first dichroic mirror, i.e., dichroic mirror 1, can be selected to transmit the fluorescence induced to it from the plurality of fluorescence from the first dichroic mirror to the corresponding imaging device, and to reflect the remaining fluorescence from the plurality of fluorescence incident on it to the next adjacent second dichroic mirror, i.e., dichroic mirror 2, and of the plurality of second dichroic mirrors, the last second dichroic mirror A dichroic mirror, i.e., the dichroic mirror i, is selected to guide the fluorescence incident thereon to the corresponding imaging device by reflection. For each of the multiple second dichroic mirrors other than the second dichroic mirror closest to the first dichroic mirror and the final second dichroic mirror, for example, the dichroic mirror 2 is selected to reflect the fluorescence guided thereon to the corresponding imaging device and transmit the remaining fluorescence of the at least one fluorescence incident thereon to the next adjacent second dichroic mirror.

[0039] Specifically, as another example, referring to an example of the fluorescence induction device 150'' shown in Figure 1c, the fluorescence induction device 150'' includes a plurality of second dichroic mirrors arranged in the order of dichroic mirror 1, dichroic mirror 2, ..., dichroic mirror i. Of the plurality of second dichroic mirrors, the second dichroic mirror located closest to the first dichroic mirror, i.e., dichroic mirror 1, is selected to reflect the fluorescence induced to it from the plurality of fluorescence from the first dichroic mirror to the corresponding imaging device, and to transmit the remaining fluorescence from the plurality of fluorescence incident to the adjacent next second dichroic mirror, i.e., dichroic mirror 2, and of the plurality of second dichroic mirrors, the last second dichroic A dichroic mirror, i.e., the dichroic mirror i, is selected to guide the fluorescence incident thereon to the corresponding imaging device by reflection. For each of the multiple second dichroic mirrors other than the second dichroic mirror closest to the first dichroic mirror and the final second dichroic mirror, for example, the dichroic mirror 2 is selected to reflect the fluorescence induced thereon to the corresponding imaging device and transmit the remaining fluorescence of the at least one fluorescence incident thereon to the next adjacent second dichroic mirror.

[0040] A dichroic mirror may be positioned such that the light it receives is incident on it at a certain range of incident angles, which facilitates the reflection and / or transmission of the light received by the dichroic mirror, particularly in the case of mixed light. The positioning of the dichroic mirror may be determined by considering the upstream components that transmit light to the dichroic mirror. The range may be an angular range including, for example, 45 degrees. Different dichroic mirrors may have different incident angle ranges as required by design.

[0041] Each of the plurality of imaging devices is positioned to receive the corresponding fluorescence from the plurality of fluorescence induced by the fluorescence induction device, and to image the fluorescence to obtain fluorescence information corresponding to the fluorescence to be detected. The imaging devices may be implemented in various ways. According to one possible embodiment, each of the plurality of imaging devices includes an optical filter, an imaging lens, and a camera arranged in a series, wherein for each imaging device, the optical filter filters the fluorescence induced to the imaging device by the fluorescence induction device, transmits the filtered fluorescence to the imaging lens, the imaging lens focuses the filtered fluorescence transmitted through the optical filter to the camera, and images it with the camera to obtain fluorescence information corresponding to the fluorescence to be detected. For each imaging device including the lens and camera, its center may be kept to coincide with the transmission / reflection center of the corresponding element in the fluorescence induction device, for example, a second dichroic mirror that induces the corresponding fluorescence to the imaging device. The camera may be a TDI camera suitable for TDI imaging.

[0042] Multiple lasers of different wavelengths may be generated by at least one laser source, and the optical fiber may be positioned to receive the multiple lasers of different wavelengths from the at least one laser source, further form the multiple lasers of different wavelengths, and transmit the multiple lasers of different wavelengths to the beam splitting device to realize the multiple incident lasers of different wavelengths. The at least one laser source may be included in or outside the detection device.

[0043] Advantageously, the at least one laser source includes a plurality of laser sources, each of which can be used to generate a plurality of lasers of different wavelengths.

[0044] In the embodiment shown in Figure 1, the beam splitting device 120 may be advantageously selected such that the angle between the emission directions of adjacent wavelength excitation lasers among the plurality of excitation lasers emitted from the beam splitting device is greater than or equal to a threshold angle, and the difference between the incidence angles of adjacent wavelength excitation lasers incident on the first dichroic mirror is greater than or equal to a desired angular difference. The desired angular difference may be determined based on the ratio of the minimum spot spacing to the focal length of the objective lens, for example, greater than or equal to the ratio of the minimum spot spacing to the focal length of the objective lens. The minimum spot spacing indicates the minimum distance between focused spots that need to be separated from each other and are formed on the sample by the plurality of excitation lasers, and may be appropriately determined in various ways depending on the situation. For example, the minimum spot spacing may be related to the requirements of a particular application, or may be predetermined based on a particular application targeted by the detection device. The required minimum spacing between spots may vary for different detection scenarios, detection objectives, and / or samples to be detected. For example, the minimum spot spacing may be determined such that it is greater than the size of the spot formed on the surface of the sample by the multiple coupled lasers from the optical fiber without dispersing the multiple lasers (the size of the narrow side of the spot, i.e., the width of the spot; in Figure 1, the lateral size of the illustrated spot), and the corresponding direction is the direction along which the spacing between the separated spots lies. When this condition is met, it is advantageous for the minimum spot spacing to be as small as possible. If the minimum spot spacing is relatively large, the focused spot and part of the image may exceed the actual field of view of the objective lens, which may cause image degradation. For example, in some cases, the minimum spot spacing may be determined to be equal to the height of the actual field of view of the imaging system on the sample for a particular application. As a simplified method, the threshold angle may be determined based on the ratio of the minimum spot spacing to the focal length of the objective lens.For example, the threshold angle may be determined to be equal to the ratio of the minimum spot spacing to the focal length of the objective lens multiplied by a coefficient, the coefficient of which may be predetermined to take into account, for example, whether the excitation laser emitted from the beam splitting device needs to be shaped / scaled before being received by the first dichroic mirror, or the angular magnification that the excitation laser emitted from the beam splitting device needs to receive before being received by the first dichroic mirror, etc. For example, the coefficient may be equal to 1 if, for example, there is no shaping device between the beam splitting device and the first dichroic mirror. As another example, if a shaping device is provided between the beam splitting device and the first dichroic mirror, the coefficient may be determined, for example, based on the angular magnification of the shaping device, and is equal to the reciprocal of the absolute value of the angular magnification.

[0045] The detection device may optionally include at least one of the following beam shaping devices: a first beam shaping device positioned between the optical fiber and the beam splitting device, which is configured to shape the multiple different wavelength incident lasers emitted from the optical fiber and to transmit the shaped incident lasers to the beam splitting device; and a second beam shaping device positioned between the beam splitting device and the first dichroic mirror, which is configured to shape the multiple excitation lasers emitted from the beam splitting device corresponding to the multiple different wavelengths of the incident lasers and to transmit the shaped multiple excitation lasers to the first dichroic mirror. Each of the first beam shaping device and the second beam shaping device may be implemented in various ways. For example, the first beam shaping apparatus includes, but is not limited to, a first lens group that performs desired shaping and scaling of the incident laser incident on the beam splitting apparatus, for example, the incident laser may be incident on the beam splitting apparatus to form a spot of a desired shape and / or size, and the second beam shaping apparatus includes, but is not limited to, a second lens group that performs desired shaping and scaling of the excitation laser emitted from the beam splitting apparatus, for example, the excitation laser may be incident on the first dichroic mirror to form a spot of a desired shape and / or size. Furthermore, the angular magnification of each of the first beam shaping apparatus and the second beam shaping apparatus can be appropriately determined depending on the situation, and its specific configuration and structure can be designed based on the desired angular magnification.

[0046] According to one possible embodiment, the beam splitting device includes a monodisperse prism, which is positioned such that each of the multiple different wavelength incident lasers from the optical fiber is incident on a first refractive surface of the monodisperse prism, and each of the multiple excitation rays then exits from a second refractive surface of the monodisperse prism that is different from the first refractive surface. The angles of incidence at which the multiple different wavelength incident lasers are incident on the first refractive surface of the monodisperse prism may be different, not fixed, and may be related to the orientation and positioning angle of the monodisperse prism. Advantageously, the monodisperse prism may be positioned such that the multiple different wavelength incident lasers are incident on the first refractive surface of the monodisperse prism at a predetermined angle of incidence, the predetermined angle of incidence being selected such that the deviation angle between the incident direction of the incident laser incident on the first refractive surface and the exit direction of the excitation laser corresponding to the incident light and exiting from the second refractive surface is minimized. The monodispersion prism may have an apex angle formed between the first refractive surface and the second refractive surface, where the first refractive surface and the second refractive surface are adjacent surfaces of the monodispersion prism, and the monodispersion prism is selected such that the narrow angle can be greater than or equal to the threshold angle by a combination of the apex angle of the monodispersion prism and the material selected to form the monodispersion prism. The monodispersion prism is not particularly limited, but may be, for example, a triangular prism, a right-angle prism with an apex angle of 45 degrees made of N-SF11 material, a square prism, etc. For example, when using a square prism, a certain angle of the square prism may be used as the apex angle, and the two adjacent refractive surfaces of the square prism defining this angle can be used to refract the laser as an incident surface for receiving the laser and an output surface for emitting the laser, respectively, and these can be realized by appropriately arranging the square prism. For a given monodispersion prism, its material, apex angle, etc., are known, and the given angle of incidence may be determined by related technical means such as a table index or calculation.Furthermore, for a given monodisperse prism, the dispersion capability of the prism can be calculated for light of any wavelength incident on the prism at such a predetermined incident angle using formulas or algorithms available in the relevant art (e.g., "New Concept Physics Course-Optics" by Kaihua ZHAO). As a simplified method for the solution of this disclosure, for the plurality of lasers used, light having an intermediate wavelength within the wavelength range of the plurality of lasers may be selected, and the amount of change in the propagation direction of the selected light after it has been incident on a prism that can be selected at such a predetermined incident angle and refracted by the prism, i.e., the minimum deviation angle of the selected light, may be calculated, and a prism that satisfies the condition that the minimum deviation angle of the selected light is greater than or equal to the threshold angle may be determined as a prism that can be used.

[0047] According to other possible embodiments, the beam splitting device includes a plurality of dispersion prisms. In this case, each of the plurality of dispersion prisms has an apex angle formed between its first refractive surface and a second refractive surface, and is arranged to receive the laser incident thereon at its first refractive surface and emit the laser from its second refractive surface, and the number of the plurality of dispersion prisms and the combination of the apex angle and material of each of the plurality of dispersion prisms are selected such that the narrow angle is greater than or equal to the threshold angle. When considering the number, arrangement, and relative positions of the dispersion prisms, such combinations may be determined using relevant data, formulas, and algorithms available in the relevant art, as described above for a single prism. In this case, among the plurality of dispersion prisms, the first dispersion prism that first receives the multiple different wavelengths of incident lasers from the optical fiber may be arranged so that the multiple different wavelengths of incident lasers from the optical fiber are incident on the first refractive surface of the first dispersion prism and then exit from the second refractive surface of the first dispersion prism, and each of the plurality of dispersion prisms other than the first dispersion prism may be arranged so that each laser emitted from the adjacent dispersion prism is incident on its first refractive surface and exits from its second refractive surface. Compared to using a single dispersion prism, using multiple dispersion prisms provides greater flexibility in the direction of the optical path and fewer constraints on the arrangement or angle of other components in the detection device, but the laser transmittance is lower when using multiple dispersion prisms. Therefore, for specific applications, it is possible to choose to use a single dispersion prism or multiple dispersion prisms depending on the actual needs.

[0048] As described above, the beam splitting device can also be implemented using a grating. When a grating is used, an appropriate combination of the orientation, position, and possible number of gratings may be determined by data available in the relevant technology (such as available grating types, parameters, and structures), equations (such as the basic equations of grating diffraction), algorithms, etc., so that the beam splitting device satisfies the relevant requirements described above.

[0049] In this specification, the use of the terms “forward” / “backward,” “front” / “rear,” “preceding” / “following,” and “previous” / “next” is based on the direction of light propagation. For example, when the same ray propagates through multiple components, the component that the ray first passes through during propagation may be called the forward component, preceding component, or previous component, in relation to the components that the ray passes through afterward during propagation.

[0050] Furthermore, the detection device of this disclosure is not limited to using a laser from an optical fiber as described in the above embodiments, but may also use excitation light from various other light sources, such as light emitted from an LED light source or light emitted from a halogen light source.

[0051] As shown in Figure 2, a detection device 20 according to another embodiment of the present disclosure includes a multimode optical fiber 210, a first beam shaping device 270, a laser filter 290, a dispersion prism 220, a second beam shaping device 280, a dichroic mirror 1 230, an objective lens 240, dichroic mirrors 2 to dichroic mirror i+1 and optical filter 1 to optical filter i constituting a fluorescence induction device 250, and imaging lens 1 to imaging lens i and camera 1 to camera i constituting an imaging system 260, where i represents the type / number of different wavelength lasers coupled through the multimode optical fiber and is an integer greater than 1. For the purpose of clarity and simplification, only three wavelength lasers are shown in Figure 2 to schematically represent i wavelength lasers, but it should be understood that the present disclosure is not limited to the case of three wavelength lasers and may also be applicable to more or fewer wavelength lasers.

[0052] As shown in Figure 2, the detection device uses a multimode fiber 210 to coaxially couple i lasers of different wavelengths (i≧2), then outputs the coupled laser of different wavelengths through the multimode fiber, shapes the coupled laser into an optical path using the first beam shaping device 270, filters it with the laser filter 290, and then incidents it on the first refractive surface of the dispersion prism 220. According to the law of refraction, lasers of different wavelengths have different refractive indices, i.e., different angles of refraction. Therefore, the lasers of different wavelengths have different propagation angles after being refracted by the first refractive surface of the dispersion prism, and after the lasers of different wavelengths are further refracted by the second refractive surface of the dispersion prism, the difference between the propagation angles of the lasers of different wavelengths is amplified. In this way, a narrow angle greater than zero is created between the transmission directions of the lasers of different wavelengths. In general, the shorter the wavelength of a laser, the larger its corresponding refractive index and angle of refraction. After dispersion by the dispersion prism, i lasers having different propagation angles (laser 1, laser 2, and laser i, schematically shown in Figure 2) are reflected by the dichroic mirror 1 230, focused by the objective lens 240, and then irradiated onto the surface of the sample 200. Since each laser has a different propagation angle, each laser forms its own spot (spot 1, spot 2, and spot i, schematically shown in Figure 2), irradiating at different locations on the sample, and each laser spot at the irradiated location on the sample originates from a single wavelength laser.

[0053] To clearly illustrate the operation process of the detection device shown in Figure 2, the device will be further described using an example where three lasers A, B, and C (not shown) are coupled using a multimode fiber, assuming i=3. The coupled laser formed by the three lasers A, B, and C is reflected by the dichroic mirror 1 230, focused by the objective lens 240, and then irradiated at different positions on the surface of the sample. At some point, the three lasers A, B, and C each have illumination regions a, b, and c (not shown) on the sample, respectively, and excite the corresponding fluorescence AX, BX, and CX (not shown) in regions a, b, and c. Assuming that region c is at the front, region a is at the back, and region b is in the center of the sample's direction of movement, the sample scanning direction is the same as the direction of travel of the illumination region of laser A (in this case, region a). The three lasers A, B, and C simultaneously excite fluorescence, and at each point in time, only one fluorescence is excited and emitted at each position on the illuminated sample. At the next point in time after the sample movement, laser A excites fluorescence AX' in a new region, laser B excites fluorescence BX' in region a, and laser C excites fluorescence CX' in region b. By moving back and forth in this manner, the scanning of the entire sample can be completed. During the scanning of the sample, all simultaneously excited fluorescence is transmitted through the objective lens to the dichroic mirror 1, then to the dichroic mirror 2, and guided through the dichroic mirror 2 to the dichroic mirror 4 and the optical filters 1 to 3 for filtering, and then imaged by the imaging lenses 1 to 3 and captured by the cameras 1 to 3.For example, the dichroic mirror 2 is selected to transmit one of the fluorescence AX, BX, and CX transmitted to it, for example, fluorescence AX, and guide it to the corresponding optical filter 1, and to reflect the remaining fluorescence, for example, fluorescence BX and CX, to the dichroic mirror 3; the dichroic mirror 3 is selected to reflect one of the fluorescence BX and CX transmitted to it, for example, fluorescence BX, and guide it to the corresponding optical filter 2, and to transmit the remaining fluorescence, for example, fluorescence CX, to the dichroic mirror 4; the dichroic mirror 4 is selected to reflect the fluorescence transmitted to it, for example, fluorescence CX, and guide it to the corresponding optical filter 3. Accordingly, the optical filter 1 may be selected so that only fluorescence AX passes through it to reach the corresponding imaging lens 1, the optical filter 2 may be selected so that only fluorescence BX passes through it to reach the corresponding imaging lens 2, and the optical filter 3 may be selected so that only fluorescence CX passes through it to reach the corresponding imaging lens 3. In this way, the different fluorescence excited simultaneously corresponds one-to-one with cameras 1 to 3. Furthermore, the fluorescence is excited individually at different locations on the sample, the different fluorescence is separated, and imaged by different cameras.

[0054] Assuming that n wavelength lasers are coaxially coupled using a multimode fiber, and that T is the duration of single-channel fluorescence detection for each FOV, Wi is the power output of each laser, and Si is the area of ​​the laser spot on the sample, then when the first related technique is employed, the laser irradiation power density received by a unit area of ​​the sample is

number

number

number

[0055] In the detection device shown in Figure 2, the dispersion prism may be appropriately arranged at various feasible angles and orientations. The arrangement of the dispersion prism may be determined, for example, at least in part on the dichroic mirror 1, such that the emission angle of the excitation laser emitted from the dispersion prism after refracting by the dispersion prism matches the range of incident ray angles required by the dichroic mirror 1. Further illustrative descriptions regarding the selection of the dispersion prism (e.g., prism material, apex angle, etc.) are given below. Assuming that the focal length of the objective lens in the detection device is denoted as f, and the minimum required distance between spots that need to be spaced apart from each other and formed on the sample by multiple lasers (i.e., the minimum spot spacing) is denoted as d, and that the approximation conditions "the objective lens is a thin lens and the actual field of view of the objective lens is less than 5°" are met, then the minimum required angle of adjacent lasers of wavelengths that need to be spaced apart from each other and incident on the dichroic mirror 1 is Δθ T ≈ d / f, and this angle Δθ T This is sometimes called the first threshold angle. The dispersion capability of a prism is determined by the material of the prism, the size of the apex angle (the angle between the incident and exit surfaces of the prism), etc. Taking the case where the dispersion prism is a triangular prism as an example, assuming that the laser is incident on the first refractive surface of the triangular prism in a direction that produces the minimum deviation angle, the angle Δθ is determined based on the distance d. T It is possible to calculate Δθ. T Once determined, the condition that the angle between adjacent wavelength excitation lasers emitted from the dispersion prism must satisfy is that the angle between them is the second threshold angle Δθ.T The condition that it must be greater than or equal to can be determined, for example, by considering the angle magnification of the second beam shaping device provided between the beam splitting device and the dichroic mirror 1, and the angle Δθ T And it can be determined based on the angle magnification, for example, Δθ T ' may be equal to the first threshold angle divided by the absolute value of the angle magnification. A suitable prism may be selected from available prisms based on the prism dispersion equation so that the angle between adjacent wavelength excitation lasers emitted from the prism satisfies the relevant requirements, depending on the material and apex angle of the selected prism. Specifically, from the principles of geometrical optics, when a laser is incident on the prism in a direction that produces the minimum deviation angle, the resulting laser image is curved minimally (i.e., aberrations are minimized), and for a laser incident at this angle of incidence, the angular dispersion capability D of the prism θ It is known that this can be shown by equation (1).

[0056]

number

[0057] In the formula, α represents the apex angle of the prism, n represents the refractive index of the prism (determined by the material of the prism), λ represents the wavelength of the laser, dn / dλ represents the dispersion index of the prism, b represents the length of the bottom edge of the prism, and a represents the beam width (related to a specific application, different applications require different beam widths).

[0058] The detection device 30 according to still other embodiments of the present disclosure is shown in FIG. 3. Hereinafter, regarding the detection device of the present disclosure, a case where lasers of two wavelengths are incident on the detection device shown in FIG. 3 via a coaxial coupling optical fiber, the beam shaping device includes a first illumination lens group 370 and a second illumination lens group 380, and the dispersion prism is a triangular prism 320 will be further described as an example. The first illumination lens group and the second illumination lens group are combined to form a shaping and scaling system. The dichroic mirror group 330 may correspond functionally and structurally to the combination of the first dichroic mirror and the fluorescence induction device in FIG. 1, or the dichroic mirrors 1 to the dichroic mirror i + 1 in FIG. 2. The triangular prism is a 45° dispersion prism. The optical path is located in the YZ plane, and the sample plane 300 of the sample irradiated by the laser is perpendicular to the YZ plane.

[0059] Lasers of two wavelengths, namely, a green laser and a red laser, are output via a coaxial coupling optical fiber 310. After being shaped by the first illumination lens group 370 and sequentially refracted by the first refracting surface 3201 of the prism 320 and the second refracting surface 3202 of the prism 320, the principal rays of the two lasers form an included angle therebetween. The light rays are further shaped by the second illumination lens group 380, and the final included angle between the central rays of the two lasers exiting from the second illumination lens group needs to be the above-mentioned threshold angle Δθ T as described above. The principal rays of the two lasers with different emission angles are transmitted to the objective lens 340 through the dichroic mirrors of the dichroic mirror group 330, and are condensed onto the sample by the objective lens to form an image, and spots of the two lasers are formed at different positions on the sample and are separated on the sample. On the other hand, the fluorescence excited on the sample by the laser is received by the objective lens 340, split, and guided to the imaging system 360, and is imaged by the dichroic mirrors of the dichroic mirror group 330.

[0060] The difference between the central wavelengths of the green laser and the red laser is 128 nm, and the height of the actual field of view of the imaging system on the sample is 80 μm. Here, "central wavelength" means the peak wavelength of the laser. In order to space out the laser spots on the sample and prevent interference with each other, the distance between adjacent laser spots of different wavelengths on the sample can be preset to be greater than 80 μm. Assuming that the focal length of the objective lens is known and the maximum angle of view in the optical splitting direction of the objective lens is less than 5°, i.e., that the approximation conditions are met, Δθ T =0.4°, that is, the angle |Δθ| between the central beams of adjacent wavelength lasers emitted from the second illumination lens group must be greater than 0.4°, as determined by the method described above.

[0061] In the YZ plane shown in Figure 3, the beam diameter of the laser at approximately the center position between the first illumination lens group and the second illumination lens group is approximately 13.5 mm. The distance of the center position from the first illumination lens group is related to the size of the optical fiber and the focal length of the first illumination lens group. In this embodiment, assuming that the center position is approximately 50 mm from the first illumination lens group and behind the first illumination lens group, the beam diameter is the beam diameter of the laser emitted from the first illumination lens group at approximately 50 mm behind the first illumination lens group. The second illumination lens group is, in principle, similar to a telescope, with an angular magnification of M2 = -0.7, and calculations according to the above approximation conditions (i.e., approximating the objective lens as a thin lens and assuming the field of view of the objective lens is less than 5°) lead to the conclusion that the angular difference |Δθ'| between the central rays of lasers of different wavelengths emitted from the prism and reaching the second illumination lens group must satisfy inequality (2).

[0062]

number

[0063] In this embodiment, a prism with an apex angle of 45° made of N-SF11 material is selected. Since the change in the dispersion index of most optical materials does not exceed one order of magnitude within the selected laser wavelength, the dispersion index of the prism |D θ | may be calculated for the central wavelength of 596 nm within the wavelength range of the selected red and green lasers, and the dispersion index thus calculated is used to determine whether the angular difference between the red and green lasers used when emitted from the prism meets the relevant requirements, thereby determining whether the selected prism meets the requirements. Specifically, after the table index and calculation, the following is obtained:

[0064]

number

[0065]

number

[0066]

number

[0067] Here, Δλ represents the wavelength difference between the red laser and the green laser. According to equations (4) and (5), |D θ |>|Δθ'| / Δλ, that is, the selected prism satisfies the dispersion angle requirement of this embodiment.

[0068] After determining the material and apex angle of the prism, the angle of incidence of the laser to the prism may be adjusted to satisfy the minimum deviation angle condition. In this embodiment, calculations have shown that the minimum deviation angle condition is satisfied when the angle of incidence is 43.1°. After optical simulation, the angle between the principal rays of the two wavelengths of laser after dispersion by this prism is 1.2°, and when the laser finally reaches the objective lens, the principal rays of the red laser and the green laser form an angle of 0.82° between the principal rays of the red laser and the principal rays of the green laser, i.e., an angle greater than 0.57°, thus satisfying the requirement. Finally, the spot distance between the spot formed by the red laser and the spot formed by the green laser on the sample is approximately 0.16 mm. As shown in Figure 4, the simulation results indicate that the upper band-shaped spot is spot 4001 formed by the red laser, and the lower band-shaped spot is spot 4002 formed by the green laser, with the centers of the red and green spots separated by a distance of approximately 0.16 mm.

[0069] This embodiment uses TDI imaging technology. When each laser is turned on, the corresponding camera is triggered to capture an image, and the camera integration process as the system scans the sample is shown in Figure 5. During the fluorescence excitation process, the red and green lasers excite different regions of the sample, respectively, and the excited regions of the laser spots on the sample correspond to the corresponding cameras, which have a one-to-one correspondence with respect to the object-image relationship. Assuming that the red laser corresponds to camera A and the green laser corresponds to camera B, in TDI imaging technology, the camera integration direction is along the narrow side of the spot, and the fluorescence excitation is continuous. When the lasers excite the sample, camera integration is triggered synchronously; that is, the trigger interval between the camera trigger sources corresponding to the red and green lasers is equal to the time interval required for the spot to move a distance of 0.16 mm relative to the sample (the spot is fixed, and the sample is moved driven by a slide table positioned in the XY plane). When the green laser enters the sample area, fluorescence excitation begins, and simultaneously, integration of camera B begins, while camera A remains in standby mode. When the red laser enters the sample, integration of camera A begins, while camera B is still integrating. When the green laser exits the sample, integration of camera B ends, but camera A is still integrating. When the red laser exits the sample, integration of camera A ends, and fluorescence detection for that row of the sample is completed. The time required to move the sample 0.16 mm is much shorter than the integration duration for one row, so the duration required for fluorescence detection in an entire row is approximately equal to the duration of single-channel fluorescence detection. When moving to the next row, the red and green lasers enter the sample in the reverse order and integrate in the reverse order. In this way, scanning detection of the entire sample is completed.

[0070] The disclosure may also be implemented as a detection system, for example, a gene sequencing system including a detection device as described above. In the case of such a gene sequencing system, the imaging system of the detection device of the disclosure is used to collect a fluorescence signal on a sequencing chip which is the sample to be detected.

[0071] Figure 8a shows the relationship between data quality Q30 and read length of gene detection results obtained by performing a double-ended test on a test subject, i.e., a DNA sequence, using the first related technique (simultaneous excitation), the second related technique (time-resolved excitation), and this disclosure. Figure 8b shows the relationship between detection error rate and read length of gene detection results obtained by performing a double-ended test on the same test subject, i.e., a DNA sequence, using the first related technique (simultaneous excitation), the second related technique (time-resolved excitation), and this disclosure. The first related technique, the second related technique, and this disclosure are each configured for gene detection under the same conditions. In Figures 8a and 8b, dashed lines indicate detection results obtained using the first related technique, hollow lines ("time-resolved excitation 1") indicate detection results obtained using the second related technique, and solid lines ("this disclosure") indicate detection results obtained using this disclosure. To compare the technical effects of the first related technology, the second related technology, and the present disclosure, the Q30 and the detection error rate (i.e., the error rate in identifying bases) in the gene detection results are used as evaluation indicators, with higher Q30 and lower detection error rate indicating better and more accurate detection results. As can be seen from Figures 8a and 8b, the difference in initial results between the two related technologies and the present disclosure at shorter read lengths is very small, but in the first related technology, both Q30 and the detection error rate deteriorate as the read length increases (generally characterized by a gradual decrease in Q30 and a gradual increase in the detection error rate), while in the second related technology and the present disclosure, the degree of deterioration of Q30 and the detection error rate is smaller than in the first related technology, and in addition, in both the present disclosure and the second related technology, both Q30 and the detection error rate are very similar overall, being the same or substantially the same at some read lengths. Therefore, as far as the results of gene sequencing are concerned, the Second Related Technique and the present disclosure are similar and both are significantly better than the First Related Technique, however the Second Related Technique requires twice as much time to image compared to the First Related Technique, while the detection duration required in the present disclosure is the same as in the First Related Technique.

[0072] The solutions of this disclosure can be applied to a variety of applications requiring fluorescence excitation and fluorescence detection, and are particularly suitable for biochemical detection such as gene detection or other cases that require exciting a sample to generate fluorescence and then detecting the excited fluorescence.

[0073] The technical means used by this disclosure to achieve its intended purpose and effects should be understood more deeply and specifically by the detailed description of specific embodiments. However, the accompanying drawings are for reference and illustrative purposes only and are not intended to limit this disclosure.

Claims

1. The imaging system includes a beam splitting device, a first dichroic mirror, an objective lens, a fluorescence induction device, and a plurality of imaging devices. The beam splitting device receives and splits incident light rays of multiple different wavelengths from an optical fiber, forming multiple excitation rays that are emitted from the beam splitting device in different exit directions corresponding to the multiple different wavelengths of the incident light rays, and each of the excitation rays is configured to have a wavelength that corresponds one-to-one with the multiple different wavelengths of the incident light rays. The first dichroic mirror is arranged to receive the plurality of excitation rays emitted from the beam splitting device, which correspond one-to-one with the plurality of different wavelengths of the incident light, transmit the plurality of excitation rays to the objective lens, which focuses the plurality of excitation rays through the objective lens onto a plurality of different regions of the sample to be detected, which excites a plurality of fluorescence molecules in the plurality of different regions of the sample where the plurality of excitation rays are focused, receive the plurality of fluorescence molecules excited by the plurality of excitation rays, and transmit the plurality of fluorescence molecules to the fluorescence induction device. The objective lens is arranged to receive the plurality of excitation rays transmitted by the first dichroic mirror, to focus the plurality of excitation rays onto the plurality of different regions of the sample, and to transmit the plurality of fluorescence excited by the plurality of excitation rays to the first dichroic mirror. The fluorescence induction device is configured to receive the plurality of fluorescence transmitted by the first dichroic mirror, to guide the plurality of fluorescence to the plurality of imaging devices, and to image each of the plurality of fluorescence by one of the plurality of imaging devices corresponding to the fluorescence. Each of the plurality of imaging devices is arranged to receive one of the plurality of fluorescence induced by the fluorescence induction device, image the fluorescence, and obtain fluorescence information corresponding to the fluorescence to be detected. The beam splitting device is configured such that, among the plurality of excitation rays emitted from the beam splitting device, the angle between the emission directions of adjacent excitation rays is greater than or equal to a threshold angle. The threshold angle is based on the ratio of a predetermined minimum interval to the focal length of the objective lens. A detection device in which the predetermined minimum interval indicates the minimum distance between focal spots that need to be spaced apart from each other and formed on the sample by the plurality of excitation rays.

2. Furthermore, it includes at least one light source configured to generate multiple excitation lights of different wavelengths, The optical fiber is arranged to receive multiple excitation light beams of different wavelengths from at least one light source, form multiple incident light beams of different wavelengths, and transmit the multiple incident light beams of different wavelengths to the beam splitting device. The detection device according to claim 1.

3. moreover, A first beam shaping device is positioned between the optical fiber and the beam splitting device, and is configured to shape the multiple incident light rays of different wavelengths emitted from the optical fiber and to transmit the shaped incident light rays to the beam splitting device. The system includes at least one of the following: a beam splitting device and a second beam shaping device, which is positioned between the beam splitting device and the first dichroic mirror, and is configured to shape the plurality of excitation rays emitted from the beam splitting device in a one-to-one correspondence with the plurality of incident light of different wavelengths, and to transmit the shaped plurality of excitation rays to the first dichroic mirror. The detection device according to claim 1.

4. The second beam shaping device has an angle magnification, If the detection device includes the second beam shaping device, the threshold angle is equal to the product of the ratio of the predetermined minimum interval to the focal length of the objective lens and the reciprocal of the absolute value of the angle magnification. The detection device according to claim 3.

5. The first beam shaping apparatus includes a first lens group, The second beam shaping apparatus includes a second lens group, The detection device according to claim 3.

6. The beam splitting device includes at least one dispersion prism, or The beam splitting apparatus includes at least one grid, The detection device according to claim 1.

7. The at least one dispersion prism includes a monodispersion prism, The aforementioned monodisperse prism, The multiple incident light rays of different wavelengths from the optical fiber are incident on the first refractive surface of the monodisperse prism. Subsequently, the plurality of excitation rays are arranged such that each of them exits from a second refractive surface of the monodisperse prism that is different from the first refractive surface. The detection device according to claim 6.

8. The aforementioned multiple incident light rays of different wavelengths are incident on the first refractive surface of the monodisperse prism at a predetermined incident angle, The predetermined incident angle is selected such that the deviation angle between the incident direction of the incident light ray incident on the first refractive surface and the exit direction of the plurality of excitation light rays corresponding to the incident light ray and emitted from the second refractive surface is minimized. The detection device according to claim 7.

9. The first refractive surface and the second refractive surface are adjacent surfaces of the monodisperse prism, The monodispersion prism has an apex angle formed between the first refractive surface and the second refractive surface, The monodispersion prism is selected such that the apex angle of the monodispersion prism and the material selected to form the monodispersion prism can make the narrow angle greater than or equal to the threshold angle. The detection device according to claim 7.

10. The monodispersion prism is a right-angle prism with a vertex angle of 45 degrees made of N-SF11 material, or The aforementioned monodispersion prism is a triangular prism. The detection device according to claim 9.

11. The at least one dispersion prism includes a plurality of dispersion prisms arranged in a continuous manner, Each of the plurality of dispersion prisms is arranged such that it has an apex angle formed between the first and second refractive surfaces of adjacent dispersion prisms, and that it receives each light incident on the dispersion prism at the first refractive surface of the dispersion prism and emits the light from the second refractive surface of the dispersion prism. The number of the plurality of dispersion prisms and the combination of the vertex angle and material of each of the plurality of dispersion prisms are selected such that the narrow angle is greater than or equal to the threshold angle. The detection device according to claim 6.

12. Among the plurality of dispersion prisms, the first dispersion prism that first receives the multiple different wavelengths of incident light rays from the optical fiber is arranged such that the multiple different wavelengths of incident light rays from the optical fiber enter the first refractive surface of the first dispersion prism and then exit from the second refractive surface of the first dispersion prism. With respect to each of the plurality of dispersion prisms other than the first dispersion prism, the dispersion prism is arranged such that each of the light emitted from the dispersion prism immediately preceding it is incident on the first refractive surface of the dispersion prism and exits from the second refractive surface of the dispersion prism. The detection device according to claim 11.

13. The fluorescence induction device includes a plurality of second dichroic mirrors arranged in a continuous manner. The plurality of second dichroic mirrors include a final second dichroic mirror spaced apart from the first dichroic mirror, and at least one preceding second dichroic mirror positioned between the final second dichroic mirror and the first dichroic mirror, and the plurality of fluorescence received by the first dichroic mirror is sequentially propagated through the plurality of second dichroic mirrors. Each of the preceding second dichroic mirrors is arranged such that it guides one of the plurality of fluorescence fluoresces, of which at least one fluorescence fluorescein incident on the preceding second dichroic mirror, to the imaging device corresponding to the preceding second dichroic mirror, and guides the remaining fluorescence of the at least one fluorescence fluorescein to the next second dichroic mirror adjacent to the preceding second dichroic mirror. The final second dichroic mirror is positioned to guide the fluorescence incident on the final second dichroic mirror to the imaging device corresponding to the final second dichroic mirror. The detection device according to any one of claims 1 to 12.

14. Of the at least one preceding second dichroic mirror, the second dichroic mirror closest to the first dichroic mirror is selected to transmit the fluorescence induced by the second dichroic mirror to the imaging device corresponding to the second dichroic mirror, and to reflect the remaining fluorescence of the plurality of fluorescence incident on the second dichroic mirror to the next second dichroic mirror adjacent to the second dichroic mirror, and the final second dichroic mirror is selected to guide the fluorescence incident on the final second dichroic mirror to the imaging device corresponding to the final second dichroic mirror by reflection, For each of the plurality of second dichroic mirrors other than the second dichroic mirror closest to the first dichroic mirror and the last second dichroic mirror, the second dichroic mirror is selected to reflect the fluorescence induced in the second dichroic mirror to the imaging device corresponding to the second dichroic mirror, and to transmit the remaining fluorescence of at least one fluorescence incident on the second dichroic mirror to the next second dichroic mirror adjacent to the second dichroic mirror, or Of the at least one preceding second dichroic mirror, the second dichroic mirror closest to the first dichroic mirror is selected to reflect the fluorescence induced by the second dichroic mirror to the imaging device corresponding to the second dichroic mirror, and to transmit the remaining fluorescence of the plurality of fluorescence incident on the second dichroic mirror to the next second dichroic mirror adjacent to the second dichroic mirror. The final second dichroic mirror is selected to guide the fluorescence incident on the final second dichroic mirror to the imaging device corresponding to the final second dichroic mirror by reflection. For each of the plurality of second dichroic mirrors other than the second dichroic mirror closest to the first dichroic mirror and the last second dichroic mirror, the second dichroic mirror is selected to reflect the fluorescence induced in the second dichroic mirror to the imaging device corresponding to the second dichroic mirror, and to transmit the remaining fluorescence of at least one fluorescence incident on the second dichroic mirror to the next second dichroic mirror adjacent to the second dichroic mirror. The detection device according to claim 13.

15. Each imaging device includes an optical filter, an imaging lens, and a camera arranged in a continuous manner. Regarding each of the aforementioned imaging devices, The optical filter of the imaging device filters the fluorescence induced in the imaging device by the fluorescence induction device, and then transmits the filtered fluorescence to the imaging lens of the imaging device. The imaging lens of the imaging device focuses the filtered fluorescence transmitted through the optical filter of the imaging device onto the camera of the imaging device, captures an image with the camera of the imaging device, and acquires fluorescence information corresponding to the fluorescence for detection. The detection device according to any one of claims 1 to 14.

16. The optical fiber is a single optical fiber, The single optical fiber is a coaxially coupled optical fiber, The aforementioned sample is a biological sample or a chemical sample, and The aforementioned detection device is a gene detection device. The detection device according to any one of claims 1 to 15.

17. The system includes an imaging system for collecting fluorescence signals on a sequencing chip, and an optical system positioned between the imaging system and the sequencing chip. The aforementioned optical system, A light source configured to emit incident light rays of multiple different wavelengths, A beam splitting device is configured to receive and split the multiple different wavelengths of incident light rays from the light source, to form multiple excitation rays that are emitted from the beam splitting device in different emission directions corresponding to the multiple different wavelengths of the incident light rays, and each of the excitation rays has a wavelength that corresponds one-to-one with the multiple different wavelengths of the incident light rays. A first dichroic mirror configured to receive the plurality of excitation rays emitted from the beam splitting device, which correspond one-to-one with the plurality of different wavelengths of the incident light, An objective lens is positioned between the sequence determination chip and the first dichroic mirror, and is configured to receive the plurality of excitation rays transmitted by the first dichroic mirror, focus the plurality of excitation rays onto a plurality of different regions on the sequence determination chip, excite a plurality of fluorescence molecules in each of the plurality of different regions in a one-to-one correspondence with the plurality of excitation rays, and transmit the plurality of fluorescence molecules to the first dichroic mirror. The device includes a fluorescence induction device configured to receive the plurality of fluorescence transmitted via the first dichroic mirror and to guide each of the plurality of fluorescence to the imaging system, The aforementioned imaging system is: The system includes a plurality of imaging devices that correspond one-to-one with the plurality of fluorescence sources, Each of the plurality of imaging devices is configured to receive light from one of the plurality of fluorescence induced by the fluorescence induction device that corresponds to the imaging device, and to image the fluorescence to obtain fluorescence information corresponding to the fluorescence. The beam splitting device is configured such that, among the plurality of excitation rays emitted from the beam splitting device, the angle between the emission directions of adjacent excitation rays is greater than or equal to a threshold angle. The threshold angle is based on the ratio of a predetermined minimum interval to the focal length of the objective lens. The predetermined minimum interval indicates the minimum distance between the focal spots that need to be spaced apart from each other and formed on the array determination chip by the plurality of excitation rays. Gene sequencing system.

18. A process of emitting incident light rays of multiple different wavelengths using a light source or photoconducting device, A beam splitting device receives and splits incident light rays of multiple different wavelengths from the light source or the photoconducting device, and forms multiple excitation rays that correspond to the multiple different wavelengths of the incident light rays and are emitted from the beam splitting device in different emission directions, wherein each of the excitation rays has a wavelength that corresponds one-to-one with the multiple different wavelengths of the incident light rays. The first dichroic mirror receives the plurality of excitation rays emitted from the beam splitting device, which correspond one-to-one with the plurality of different wavelengths of the incident light. The process involves: receiving the plurality of excitation rays transmitted by the first dichroic mirror using an objective lens positioned between the sample to be detected and the first dichroic mirror; focusing the plurality of excitation rays onto a plurality of different regions on the sample using the objective lens to excite a plurality of fluorescence molecules in each of the plurality of different regions in a one-to-one correspondence with the plurality of excitation rays; and transmitting the plurality of fluorescence molecules to the first dichroic mirror using the objective lens. The process includes the steps of: receiving the plurality of fluorescence transmitted by the first dichroic mirror using a fluorescence induction device; inducing the plurality of fluorescence to a plurality of imaging devices that correspond one-to-one with the plurality of fluorescence using the fluorescence induction device; receiving each of the plurality of fluorescence by one of the plurality of imaging devices corresponding to the fluorescence and imaging it; and obtaining fluorescence information corresponding to the fluorescence to be detected. The beam splitting device is configured such that, among the plurality of excitation rays emitted from the beam splitting device, the angle between the emission directions of adjacent excitation rays is greater than or equal to a threshold angle. The threshold angle is based on the ratio of a predetermined minimum interval to the focal length of the objective lens. The predetermined minimum interval indicates the minimum distance between the focal spots that need to be spaced apart from each other and formed on the sample by the plurality of excitation rays. Detection method.

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