Optical chips for exciting fluorescence signals and fluorescence detection methods

Abstract

This invention provides an optical chip for exciting fluorescence signals, comprising: a fluorescence excitation structure, a polarization rotation reflection module, and a substrate; the fluorescence excitation structure is formed on the substrate and includes a transmission waveguide and a sample well; the transmission waveguide outputs a first excitation light to the polarization rotation reflection module; the polarization rotation reflection module is used to polarize and rotate the first excitation light by 90 degrees to generate a second excitation light, and transmits the second excitation light to the transmission waveguide. This invention also provides a fluorescence detection method, wherein a first excitation light is emitted into the transmission waveguide, and a sample is placed within the evanescent field of the light transmitted through the transmission waveguide; the first excitation light is polarized and rotated by 90 degrees to generate a second excitation light, and the second excitation light is transmitted back to the transmission waveguide; based on the first and second excitation light, the fluorescence signal excited by the sample in the sample well is detected. Therefore, this invention superimposes two excitation lights with mutually orthogonal polarization states, improving the signal-to-noise ratio of the fluorescence signal without generating standing waves.

Description

Technical Field

[0001] This invention relates to the field of optical chips and optical signal detection, and in particular to an optical chip for exciting fluorescence signals and a fluorescence detection method. Background Technology

[0002] Biochip technology is a comprehensive high-tech field involving biology, chemistry, medicine, precision manufacturing, optics, microelectronics, and informatics. It is a highly interdisciplinary and popular research topic. In recent years, the excitation and collection of biofluorescence signals through optically integrated chips has gradually demonstrated significant value and potential in the field of fluorescence molecule detection, especially in the field of single-molecule fluorescence detection.

[0003] Biochip technology, through semiconductor manufacturing processes, achieves a monolithic integrated solution encompassing fluorescence signal excitation, collection, and detection. By massively generating nanoscale sample confinement sites on the chip surface and combining this with biochemical modification methods, biochip technology achieves spatial confinement at the single-molecule scale. Furthermore, compared to traditional optical systems for excitation and collection of fluorescence signals, biochip technology utilizes evanescent wave excitation via optical waveguides to achieve simultaneous excitation of a large number of sites. Simultaneously, its on-chip collection structure aligns sample confinement sites with detection pixels, enabling simultaneous collection of fluorescence signals from a large number of sites. Ultimately, biochip technology enables high-throughput fluorescence detection.

[0004] However, the effectiveness of biochip technology is highly dependent on the signal-to-noise ratio of fluorescence detection, and the single-molecule-level fluorescence signal required for biochip technology is extremely weak.

[0005] Therefore, how to provide an optical chip for exciting fluorescence signals and a fluorescence detection method to enhance the signal-to-noise ratio of fluorescence signals has become one of the technical problems that urgently need to be solved by those skilled in the art.

[0006] It should be noted that the above description of the technical background is only for the purpose of providing a clear and complete explanation of the technical solutions of the present invention and facilitating understanding by those skilled in the art. It should not be assumed that the above technical solutions are known to those skilled in the art simply because they have been described in the background section of this invention. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an optical chip for exciting fluorescence signals and a fluorescence detection method, so as to solve the problems of high power consumption and limited improvement space in improving fluorescence signal-to-noise ratio in the prior art.

[0008] To achieve the above and other related objectives, the present invention provides an optical chip for exciting fluorescence signals. The optical chip for exciting fluorescence signals includes at least: a fluorescence excitation structure, a polarization rotation reflection module, and a substrate. The fluorescence excitation structure is formed on the substrate and includes a plurality of corresponding transmission waveguides and a plurality of sample well units. The transmission waveguides are used to output a first excitation light to the polarization rotation reflection module. Each sample well unit includes one or more sample wells, which are sequentially arranged along the extension direction of the corresponding transmission waveguide and located within the evanescent field of the light transmitted by the corresponding transmission waveguide, for placing samples. The polarization rotation reflection module is connected to the output end of the transmission waveguide and is used to receive and polarize the first excitation light by 90 degrees to generate a second excitation light, and to transmit the second excitation light into the transmission waveguide.

[0009] Optionally, the optical chip for exciting the fluorescence signal further includes a light source; the light source is integrated on the substrate and connected to the input end of the transmission waveguide for generating the first excitation light.

[0010] Optionally, the optical chip for exciting the fluorescence signal further includes a second coupler; the second coupler is formed on the substrate and connected to the input end of the transmission waveguide, for coupling the first excitation light generated by an off-chip light source into the transmission waveguide.

[0011] Alternatively, when the number of transmission waveguides is greater than or equal to 2, a beam splitting structure is further provided on the substrate; the beam splitting structure is disposed between the light source and the input end of the transmission waveguide, with the common end connected to the light source and each branch end connected to the input end of each transmission waveguide.

[0012] Optionally, the polarization rotation and reflection module includes a polarization rotator and a reflector; the polarization rotator is connected between the output end of the transmission waveguide and the reflector, and is used to polarize and rotate the first excitation light by 45 degrees to generate a third excitation light and transmit the third excitation light to the reflector, and to polarize and rotate the third excitation light by 45 degrees to generate a second excitation light and transmit the second excitation light to the transmission waveguide; the reflector is used to reflect the third excitation light into the polarization rotator.

[0013] Alternatively, both the polarization rotator and the reflector are formed outside the substrate; a first coupler is also disposed on the substrate, the first coupler being an end-face coupler and connected between the output end of the transmission waveguide and the polarization rotator.

[0014] Alternatively, both the polarization rotator and the reflector are formed on the substrate.

[0015] Alternatively, the polarization rotator is a partially shallowly etched straight waveguide.

[0016] Alternatively, the reflector is a waveguide-type distributed Bragg reflector.

[0017] To achieve the above and other related objectives, the present invention also provides a fluorescence detection method, which includes at least the following steps: S1: emitting a first excitation light and transmitting it to a transmission waveguide; placing a sample within the evanescent field range of the light transmitted by the transmission waveguide; S2: rotating the first excitation light output from the transmission waveguide by 90 degrees to generate a second excitation light; transmitting the second excitation light to the transmission waveguide; S3: detecting a fluorescence signal; wherein the fluorescence signal is generated by exciting the sample after the first excitation light and the second excitation light are superimposed in the transmission waveguide.

[0018] As described above, the optical chip for exciting fluorescence signals and the fluorescence detection method of the present invention have the following beneficial effects:

[0019] 1. The present invention performs a 90-degree polarization rotation on the first excitation light to generate a second excitation light. The second excitation light is reflected back into the transmission waveguide and superimposed with the first excitation light in the transmission waveguide. Without generating standing waves or increasing chip power consumption, the intensity of the excitation light is increased, thereby improving the signal-to-noise ratio of the fluorescence signal.

[0020] 2. By integrating the light source, fluorescence excitation structure, polarization rotator and reflector on the same substrate, this invention can improve the integration of optical chips and facilitate the industrial production of optical chips. Attached Figure Description

[0021] Figure 1 The diagram shown is a schematic diagram of the first structure of the optical chip for exciting fluorescence signals according to the present invention.

[0022] Figure 2 The image shown is a cross-sectional view of the fluorescence excitation structure of the present invention.

[0023] Figure 3 The image shown is a top view of the excitation fluorescence signal of the present invention.

[0024] Figure 4 The diagram shows the excitation light enhancement effect of the optical chip for exciting fluorescence signals according to the present invention.

[0025] Figure 5 The diagram shows a second structural schematic of the optical chip for exciting fluorescence signals according to the present invention.

[0026] Figure 6 The diagram shown is a third structural schematic of the optical chip for exciting fluorescence signals according to the present invention.

[0027] Figure 7 The image shown is a top view of a portion of the structure of the optical chip for exciting fluorescence signals according to the present invention.

[0028] Figure 8 The diagram shown is a partial cross-sectional view of the optical chip for exciting fluorescence signals according to the present invention.

[0029] Figure 9 The diagram shown is a schematic diagram of the fourth structure of the optical chip for exciting fluorescence signals according to the present invention.

[0030] Figure 10 The diagram shown is a fifth structural schematic of the optical chip for exciting fluorescence signals according to the present invention.

[0031] Figure 11 This is a schematic diagram of the sixth structure of the optical chip for exciting fluorescence signals according to the present invention.

[0032] Figure 12 This diagram illustrates the intensity of excitation light in a transmission waveguide of the prior art.

[0033] Figure 13 The diagram shows the intensity of the excitation light superimposed in the transmission waveguide of this invention.

[0034] Figure 14 The diagram shown is a flowchart of the fluorescence detection method of the present invention.

[0035] Component designation explanation

[0036] 1. Optical chip for exciting fluorescence signals

[0037] 1a light source

[0038] 1b fluorescence excitation structure

[0039] 11 Transmission Waveguide

[0040] 12 sample wells

[0041] 1c polarization rotator

[0042] 1D reflector

[0043] 1e substrate

[0044] 1f beam splitting structure

[0045] 1g second coupler

[0046] 1h polarization rotation reflection module

[0047] 1i First Coupler Detailed Implementation

[0048] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0049] Please see Figures 1-14 It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the illustrations only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0050] The most direct ways to improve the signal-to-noise ratio of optical signals are to increase the input optical power, reduce chip link loss, and enhance the excitation light field. Increasing the input optical power requires increasing the power of the excitation light source or increasing the number of excitation light sources, which will increase costs. Reducing link loss requires increasing the loss of chip devices, and there is limited room for improvement when the product enters a relatively mature stage. However, there is still great potential to improve the signal-to-noise ratio by enhancing the excitation light field.

[0051] One approach utilizes reflectors to form standing wave cavities, enhancing the light intensity within the transmission waveguide on the chip. However, the light intensity fluctuations within the standing wave within the waveguide are significant, leading to uneven excitation intensity across the detector array and resulting in substantial detection errors. Even with optimized detector design, manufacturing process errors make this effect unavoidable.

[0052] Therefore, in order to increase the light intensity within the waveguide and eliminate the adverse effects of standing waves, this invention proposes an optical chip for exciting fluorescence signals and a fluorescence detection method, the specific technical solution of which is as follows:

[0053] Example 1

[0054] like Figure 1 As shown, this embodiment provides an optical chip 1 for exciting fluorescence signals, including: a fluorescence excitation structure 1b, a polarization rotation reflection module 1h, and a substrate 1e.

[0055] like Figure 1 As shown, the fluorescence excitation structure 1b is formed on the substrate 1e and includes a plurality of transmission waveguides 11 and a plurality of sample well units corresponding to each other; the transmission waveguides 11 are used to output the first excitation light to the polarization rotation reflection module 1h; the sample well unit includes one or more sample wells 12, which are arranged sequentially along the extension direction of the corresponding transmission waveguide 11 and located in the evanescent field of the light transmitted by the corresponding transmission waveguide 11, and are used to place the sample.

[0056] Specifically, in this embodiment, such as Figure 2 and Figure 3 As shown, a sample well unit is correspondingly disposed on a transmission waveguide 11. The sample well unit includes one or more sample wells 12, which are sequentially arranged along the extension direction of the transmission waveguide 11. Further, the sample is placed at the bottom of the sample well 12. The sample well 12 is located within the evanescent field of the light transmitted by the corresponding transmission waveguide 11, meaning the distance from the bottom of the sample well 12 to the transmission waveguide 11 is greater than zero and less than the evanescent wave penetration distance of the light transmitted by the transmission waveguide 11, facilitating the excitation of the sample by the optical signal within the transmission waveguide 11. To facilitate the subsequent acquisition of the fluorescence signal generated after the sample is excited, each sample well unit can be located above the corresponding transmission waveguide 11. As an example, firstly, each transmission waveguide 11 is formed on the substrate 1e by etching; secondly, the corresponding sample well unit is formed above each transmission waveguide 11 by deposition and etching. In practical applications, the specific process used for the fluorescence excitation structure 1b is selected as needed, and is not limited to this embodiment.

[0057] like Figure 1 As shown, the polarization rotation reflection module 1h is connected to the output end of the transmission waveguide 11, and is used to receive and polarize the first excitation light by ninety degrees to generate the second excitation light, and to transmit the second excitation light to the transmission waveguide 11.

[0058] Specifically, in this embodiment, the polarization rotation reflection module 1h functions to rotate the first excitation light by 90 degrees to generate the second excitation light, and transmits the second excitation light to the transmission waveguide 11. The first and second excitation lights are superimposed in the transmission waveguide 11, and the superimposed light signal excites the sample in the sample well 12 to generate a fluorescence signal, such as... Figure 4 As shown, since the intensity of the superimposed optical signal is increased, this embodiment can improve the signal-to-noise ratio of the fluorescence signal. For example, as... Figure 5 and Figure 6 As shown, when the polarization rotation reflection module 1h is formed on the substrate 1e, the polarization rotation reflection module 1h is directly connected to the output waveguide of the transmission waveguide 11, and the number corresponds one-to-one. As another example, such as... Figure 9 As shown, when the polarization rotation reflection module 1h is formed outside the substrate 1e, the polarization rotation reflection module 1h is connected to the output end of the transmission waveguide 11 through a coupler on the substrate 1e, and the number of modules corresponds one-to-one.

[0059] Specifically, in this embodiment, the polarization rotation and reflection module 1h includes a polarization rotator 1c and a reflector 1d. The polarization rotator 1c is disposed between the output end of the transmission waveguide 11 and the reflector 1d, and is used to polarize and rotate the first excitation light by 45 degrees to generate a third excitation light and transmit the third excitation light to the reflector 1d, and to polarize and rotate the third excitation light by 45 degrees to generate a second excitation light and transmit the second excitation light to the transmission waveguide 11. The reflector 1d is used to reflect the third excitation light into the polarization rotator 1c. Furthermore, the first excitation light and the second excitation light are superimposed in the transmission waveguide 11. Since the intensity of the superimposed optical signal can be increased, the signal-to-noise ratio of the fluorescence signal is improved.

[0060] Specifically, in this embodiment, such as Figure 5 and Figure 6 As shown, to improve the integration density of the optical chip, both the polarization rotator 1c and the reflector 1d are formed on the substrate 1e. As an example, such as... Figure 7 and Figure 8 As shown, the polarization rotator 1c is a partially shallowly etched straight waveguide, and the reflector 1d is a waveguide-type distributed Bragg reflector. Further, as... Figure 1 and Figure 9 As shown, both the polarization rotator 1c and the reflector 1d are formed outside the substrate 1e. A first coupler 1i is also disposed on the substrate 1e, connecting the output end of the transmission waveguide 11 and the polarization rotator 1c. As an example, such as... Figure 9 As shown, the polarization rotator 1c is a Faraday rotator, the reflector 1d is a high-reflection mirror, and to simplify the optical chip structure, the first coupler 1i is an end-face coupler, that is, the side of the substrate 1e is used as the light outlet to establish an optical connection between the on-chip optical device and the off-chip optical device. In practical applications, the specific types of polarization rotator 1c, reflector 1d, and first coupler 1i can be set as needed, and are not limited to this embodiment.

[0061] Specifically, in this embodiment, in order to increase the functionality and integration of the chip, such as... Figure 10 As shown, the optical chip 1 that excites the fluorescence signal also includes a light source 1a, which is integrated on the substrate 1e and connected to the input end of the transmission waveguide 11 to generate the first excitation light. Further, as... Figure 11 As shown, the optical chip 1 that excites the fluorescence signal also includes a second coupler 1g. The second coupler 1g is formed on the substrate and connected to the input end of the transmission waveguide 11, and is used to couple the first excitation light generated by the off-chip light source into the transmission waveguide 11. As an example, the second coupler 1g can be a grating coupler. In practical applications, the specific type of the second coupler 1g can be set as needed, and is not limited to this embodiment.

[0062] Specifically, in this embodiment, such as Figure 6 and Figure 9 As shown, when the number of transmission waveguides 11 is greater than or equal to 2, a beam splitting structure 1f is also provided on the substrate 1e. The beam splitting structure 1f is located between the light source 1a and the input end of the transmission waveguide 11. The common end of the beam splitting structure 1f is connected to the light source 1a, and each branch end is connected to the input end of each transmission waveguide 11. As an example, when the light source 1a is formed outside the substrate 1e, the common end of the beam splitting structure 1f is adjusted to connect to the second coupler 1g, and each branch end is still connected to the input end of each transmission waveguide 11. The branch structure 1f can be a beam splitter or a tree-like beam splitting network composed of beam splitters. In practical applications, the specific type of the beam splitting structure 1f is set as needed, and is not limited to this embodiment.

[0063] It should be noted that, in order to improve the signal-to-noise ratio of the fluorescence signal emitted by the sample, the power of the excitation source is generally increased. However, due to the large number of transmission waveguides 11 and sample wells 12 in practice, increasing the power of the excitation source would result in huge power consumption. Furthermore, without changing the original excitation source power, by reusing the remaining excitation light signal output from the transmission waveguide 11 and reflecting it back into the transmission waveguide 11 to superimpose it with the excitation light signal directly emitted from the light source 1a, the excitation light intensity can be increased, thereby increasing the signal-to-noise ratio of the fluorescence signal. Even further, since the reflected excitation light signal and the excitation light signal directly emitted from the light source 1a are essentially the same beam, this simple superposition method inevitably produces a standing wave effect, resulting in large fluctuations in light intensity within the waveguide, making it impossible to accurately excite the fluorescence signal. In summary, as... Figure 12 and Figure 13 As shown, in this embodiment, the first excitation light is polarized by 90 degrees and a second excitation light is generated, so that the first excitation light and the second excitation light are no longer coherent, suppressing the conditions for the generation of standing waves. That is, in this embodiment, superimposed light with relatively uniform light intensity can be used to excite the fluorescence signal, thereby increasing the signal-to-noise ratio of the fluorescence signal.

[0064] Example 2

[0065] like Figure 14 As shown, this embodiment provides a fluorescence detection method, including the following steps:

[0066] like Figure 14 As shown, in step S1, the first excitation light is emitted and transmitted to the transmission waveguide 11; the sample is placed within the evanescent field range of the light transmitted by the transmission waveguide 11.

[0067] Specifically, in this embodiment, the distance between the sample and the transmission waveguide 11 is greater than zero and less than the evanescent wave penetration distance of the transmission waveguide 11, so that the optical signal in the transmission waveguide 11 can excite the sample.

[0068] like Figure 14 As shown, in step S2, the first excitation light output from the transmission waveguide 11 is polarized by 90 degrees to generate the second excitation light; the second excitation light is reflected and transmitted into the transmission waveguide 11.

[0069] Specifically, in this embodiment, the first excitation light and the second excitation light are superimposed in the transmission waveguide 11. Compared to the case where they are not superimposed, as... Figure 10 and Figure 11 As shown, not only is the light intensity of the light signal greatly enhanced, but the light signal also exhibits stable performance, making it suitable for exciting the fluorescence signal of the sample.

[0070] like Figure 14 As shown, in step S3, a fluorescence signal is detected; wherein, the fluorescence signal is generated by the sample being excited by the superposition of the first excitation light and the second excitation light in the transmission waveguide 11.

[0071] Specifically, in this embodiment, since the optical signal used to excite the fluorescence signal in the transmission waveguide 11 is a superimposed first excitation light and second excitation light, the excitation light intensity is enhanced. Therefore, compared with the traditional technical solution, the signal-to-noise ratio of the fluorescence signal that can be detected in this embodiment can be greatly improved.

[0072] It should be noted that this embodiment can be implemented based on the optical chip 1 that excites the fluorescence signal in Embodiment 1, or it can be implemented based on other optical chips that are the same as or similar to the concept of this invention.

[0073] In summary, the optical chip for exciting fluorescence signals according to the present invention includes: a fluorescence excitation structure, a polarization rotation reflection module, and a substrate; the fluorescence excitation structure is formed on the substrate and includes a transmission waveguide and a sample well unit; the transmission waveguide outputs a first excitation light to the polarization rotation reflection module, and the sample well unit is arranged along the extension direction of the corresponding transmission waveguide; the polarization rotation reflection module is used to polarize and rotate the first excitation light by 90 degrees to generate a second excitation light, and transmits the second excitation light to the transmission waveguide. The fluorescence detection method of the present invention firstly emits a first excitation light into the transmission waveguide and places a sample within the evanescent field of the light transmitted through the transmission waveguide; secondly, the first excitation light is polarized and rotated by 90 degrees to generate a second excitation light, which is then transmitted back to the transmission waveguide; finally, based on the excitation light resulting from the superposition of the first and second excitation lights, the fluorescence signal generated by the excited sample is detected. Therefore, the present invention provides an integrated optical chip that superimposes two excitation lights with mutually orthogonal polarization states, enhancing the excitation light field and improving the signal-to-noise ratio of the fluorescence signal without generating standing waves or increasing chip power consumption. Therefore, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0074] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An optical chip for exciting fluorescent signals, characterized in that, The optical chip that excites the fluorescence signal includes at least: a fluorescence excitation structure, a polarization rotation reflection module, and a substrate; The fluorescence excitation structure is formed on the substrate and includes a plurality of transmission waveguides and a plurality of sample well units that correspond one-to-one; the transmission waveguides are used to output the first excitation light to the polarization rotation reflection module; the sample well unit includes one or more sample wells, which are arranged sequentially along the extension direction of the corresponding transmission waveguide and located in the evanescent field of the light transmitted by the corresponding transmission waveguide, for placing samples. The polarization rotation reflection module is connected to the output end of the transmission waveguide and is used to receive and polarize the first excitation light by 90 degrees to generate the second excitation light, and to transmit the second excitation light into the transmission waveguide.

2. The optical chip for exciting fluorescence signals according to claim 1, characterized in that: The optical chip that excites the fluorescence signal also includes a light source; the light source is integrated on the substrate and connected to the input end of the transmission waveguide to generate the first excitation light.

3. The optical chip for exciting fluorescence signals according to claim 1, characterized in that: The optical chip that excites the fluorescence signal also includes a second coupler; the second coupler is formed on the substrate and connected to the input end of the transmission waveguide, for coupling the first excitation light generated by an off-chip light source into the transmission waveguide.

4. The optical chip for exciting fluorescence signals according to claim 2 or 3, characterized in that: When the number of transmission waveguides is greater than or equal to 2, a beam splitting structure is also provided on the substrate; The beam splitting structure is disposed between the light source and the input end of the transmission waveguide, with the common end connected to the light source and each branch end connected to the input end of the respective transmission waveguide.

5. The optical chip for exciting fluorescence signals according to any one of claims 1-3, characterized in that: The polarization rotation and reflection module includes a polarization rotator and a reflector; The polarization rotator is connected between the output end of the transmission waveguide and the reflector, and is used to polarize and rotate the first excitation light by 45 degrees to generate the third excitation light and transmit the third excitation light to the reflector, and to polarize and rotate the third excitation light by 45 degrees to generate the second excitation light and transmit the second excitation light to the transmission waveguide; The reflector is used to reflect the third excitation light into the polarization rotator.

6. The optical chip for exciting fluorescence signals according to claim 5, characterized in that: Both the polarization rotator and the reflector are formed outside the substrate; a first coupler is also provided on the substrate, the first coupler being an end-face coupler, and is connected between the output end of the transmission waveguide and the polarization rotator.

7. The optical chip for exciting fluorescence signals according to claim 5, characterized in that: Both the polarization rotator and the reflector are formed on the substrate.

8. The optical chip for exciting fluorescence signals according to claim 7, characterized in that: The polarization rotator is a partially shallowly etched straight waveguide.

9. The optical chip for exciting fluorescence signals according to claim 7, characterized in that: The reflector is a waveguide-type distributed Bragg reflector.

10. A fluorescence detection method, characterized in that, The fluorescence detection method includes at least the following steps: S1: The first excitation light is emitted and transmitted into the transmission waveguide; the sample is placed within the evanescent field range of the light transmitted by the transmission waveguide; S2: The first excitation light output from the transmission waveguide is polarized by 90 degrees to generate a second excitation light; the second excitation light is then transmitted into the transmission waveguide. S3: Detect fluorescence signal; wherein the fluorescence signal is generated by the sample after the first excitation light and the second excitation light are superimposed in the transmission waveguide.

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