Method and mechanism for removing excitation fluorescence background signal interference by rotating polarization modulation

Abstract

This invention relates to a method and mechanism for removing background signal interference from excited fluorescence using rotational polarization modulation. A laser is incident by rotation, pre-processed, and then directed towards a dichroic mirror. After reflection, the laser is emitted towards the sample being tested. The stimulated fluorescence of the sample is directed towards the dichroic mirror, transmitted, and modulated with a portion of the reflected laser light to obtain DC-state stimulated fluorescence and modulated polarized laser light. After further processing, the output light is obtained, filtered, and the stimulated fluorescence information is retained while background signal interference is removed. A pre-processing unit is provided in conjunction with the laser incident channel, and a dichroic mirror is located at the end of the laser incident channel. A rotation mechanism is provided in conjunction with the laser. The sample optical detection channel on the reflected light side of the dichroic mirror has a sample position. The fluorescence optical detection channel on the transmitted light side of the dichroic mirror sequentially includes a modulation unit, a re-processing unit, and a signal processing system. This invention can be effectively distinguished by a filter; the linear polarizer can be installed at any angle, and even if the phase of the reflected background laser light is changed, it can be eliminated by filtering.

Description

Technical Field

[0001] This invention relates to the technical field of optical elements, systems or instruments, and in particular to a method and mechanism for removing interference from excitation fluorescence background signals using rotational polarization modulation. Background Technology

[0002] Fluorescent labeling has been widely used in molecular and immunoassay detection, but its detection sensitivity has consistently lagged behind that of chemiluminescence. The main limiting factor for improving excitation fluorescence sensitivity is the background interference from the excitation source. Generally, the fluorescence signal emitted under excitation is much higher than the self-emission fluorescence signal of chemiluminescence. The advantage of self-emission fluorescence lies primarily in the absence of background interference from the excitation source. If background interference from the excitation source is present, simply increasing the sensor's detection sensitivity also amplifies the interference signal proportionally. Therefore, the core challenge in improving the detection sensitivity of excitation fluorescence is eliminating the background interference introduced by the excitation source.

[0003] In existing technologies, background interference elimination primarily employs light source modulation methods. This involves generating a sequence of excitation light with specific on / off frequencies via an electrical signal switch, followed by the acquisition of fluorescence signals at the same frequency, thus separating the fluorescence signal from the ambient background light. However, the largest source of interference in the background signal is the excitation light source itself, which passes through a fluorescence filter into the detection sensor. Since the excitation light and fluorescence are at the same frequency, modulation methods cannot eliminate light source interference. Common methods for eliminating light source signal interference use narrowband filtering with fluorescence interference filters. However, due to the limited cutoff depth of the filters, the intensity of the reflected light from the excitation light source after passing through the filter determines the fluorescence signal detection sensitivity. The filter's effectiveness is further reduced when the wavelength shift between the excitation light source and the emitted fluorescence wavelength is relatively small, making it difficult to improve detection sensitivity.

[0004] To address background interference from the excitation source, one solution involves mounting two linear polarizers perpendicular to each other at 90° on the excitation and fluorescence receiving paths, respectively, to isolate the excitation source from interference. However, this solution requires absolute perpendicularity of the polarizers, posing significant challenges to fabrication and installation. Furthermore, the samples at the sample location are typically stored in containers made of organic materials such as ABS, PMMA, and PC. These materials exhibit birefringence, altering the polarization angle of the laser source. Even if the linear polarizers in the excitation and fluorescence receiving paths are perfectly perpendicular, isolation cannot be achieved. Summary of the Invention

[0005] This invention solves the problems existing in the prior art and provides a method and mechanism for removing background signal interference from excitation fluorescence using rotational polarization modulation. By using a rotating laser incident along with a modulation scheme, it achieves decoupling processing of the strictly perpendicular angle of linear polarization. Since the excitation source is rotating, modulation with a linear polarizer can obtain both DC stimulated fluorescence and modulated polarized laser with the same frequency as the rotation speed. Changes in polarization angle caused by processing and installation deviations or birefringence interference from the sample container only change the phase of the cosine wave, not the frequency, thus not affecting the filtering and isolation effect after demodulation. This invention removes background interference from the excitation source on the fluorescence signal by filtering out the modulated frequency polarized laser using a fixed-frequency filter.

[0006] The technical solution adopted in this invention is a method for removing background signal interference from excitation fluorescence using rotational polarization modulation, the method comprising the following steps:

[0007] S1 rotates the laser at a preset frequency and is incident, thus preprocessing the incident laser.

[0008] The laser pre-processed by S2 is directed at a pre-set angle to a dichroic mirror installed at a pre-set angle, typically 45°. After being reflected by the dichroic mirror, it is emitted towards the sample being tested. In fact, some of the excitation light also passes through the dichroic mirror and is emitted out. This can be addressed by special structural treatment, such as ramp treatment, so that it does not affect the normal operation of this method.

[0009] The stimulated fluorescence of the S3 detection sample is directed toward the dichroic mirror. After being transmitted through the dichroic mirror, it is modulated with part of the reflected laser to obtain stimulated fluorescence in DC state and modulated polarized laser of the same frequency.

[0010] S4 further processes the stimulated fluorescence and modulated polarized laser of S3 to obtain the output light;

[0011] S5 filters the output light, retaining the stimulated fluorescence information and removing background signal interference (invalid signal interference).

[0012] Preferably, in S1, the preprocessing includes filtering the laser light, which is itself a linearly polarized laser and a parallel light source.

[0013] Preferably, in S2, the dichroic mirror is set at 45°, with the incident surface of the dichroic mirror facing the incident laser and the detection sample.

[0014] Preferably, in step S2, the dichroic mirror reflects the laser light into the sample optical detection channel. The sample optical detection channel is equipped with a first lens, and a sample position for placing the detection sample is set at the focal length of the first lens. When the detection sample is irradiated by the excitation light, it emits fluorescence, which is stimulated fluorescence, and this fluorescence travels along the sample optical detection channel towards the dichroic mirror. Stimulated fluorescence does not have linear or circular polarization characteristics.

[0015] Preferably, in S3, the stimulated fluorescence and reflected laser of the detection sample are modulated by a linear polarizer to obtain stimulated fluorescence in DC state and modulated polarized laser (background) of the same frequency; in the modulation process, part of the reflected excitation light and stimulated fluorescence are transmitted together through a dichroic mirror and enter the fluorescence optical detection channel in parallel. After passing through the linear polarizer, the combination of stimulated fluorescence and linear polarized laser can be processed to obtain stimulated fluorescence in DC state and modulated frequency polarized laser.

[0016] The linear polarizer of the present invention can be placed at any angle in the optical path.

[0017] Preferably, in S4, the reprocessing includes filtering and focusing the stimulated fluorescence and modulated polarized laser in DC state.

[0018] Preferably, in step S5, a photosensitive sensor is used to acquire the output light signal, and the signal is filtered by a fixed-frequency filter of electronic circuit or software to retain the DC signal.

[0019] A mechanism for removing interference from excitation fluorescence background signal using rotational polarization modulation employing the method described above, the mechanism comprising a laser incident channel in conjunction with a laser, a preprocessing unit provided in conjunction with the laser incident channel, a dichroic mirror provided at the end of the laser incident channel at a preset angle, and a rotation mechanism in conjunction with the laser;

[0020] A sample optical detection channel is provided on the reflected light side of the dichroic mirror, and a sample position for placing the detection sample is provided in conjunction with the sample optical detection channel;

[0021] A fluorescence optical detection channel is provided on the transmission light side of the dichroic mirror, and a modulation unit, a reprocessing unit, and a signal processing system are sequentially provided on the fluorescence optical detection channel.

[0022] Preferably, the rotation axis of the rotating mechanism is parallel to the laser incident channel, the sample optical detection channel and the fluorescence optical detection channel are coaxial, and the laser incident channel is perpendicular to the sample optical detection channel and the fluorescence optical detection channel.

[0023] Preferably, the dichroic mirror is set at 45°, with its incident surface facing the laser incident channel and the sample optical detection channel.

[0024] This invention relates to a method and mechanism for removing background signal interference from excited fluorescence using rotational polarization modulation. In the method, a laser rotates at a preset frequency, is incident, pre-processed, and then directed at a preset angle towards a dichroic mirror installed at the same preset angle. The laser is reflected by the dichroic mirror and emitted towards the test sample. The stimulated fluorescence of the test sample is directed towards the dichroic mirror, transmitted through it, and modulated with a portion of the reflected laser to obtain DC-state stimulated fluorescence and modulated frequency polarized laser. After further processing, the output light is obtained, filtered, and the stimulated fluorescence information is retained, thus removing background signal interference. The mechanism includes a laser incident channel that works in conjunction with the laser, and is designed to work with the laser incident channel. It has a preprocessing unit, a dichroic mirror at a preset angle at the end of the laser incident channel, a rotating mechanism in conjunction with the laser, a sample optical detection channel on the reflected light side in conjunction with the dichroic mirror, and a sample position for placing the detection sample in conjunction with the sample optical detection channel, and a fluorescence optical detection channel on the transmitted light side in conjunction with the dichroic mirror, and a modulation unit, a reprocessing unit and a signal processing system in sequence in conjunction with the fluorescence optical detection channel; the signal processing system acquires the signal from the photosensitive sensor, filters out the modulated frequency polarized laser with a filter, and obtains the light intensity of the stimulated fluorescence after removing background signal interference, which can be applied to all fields of stimulated fluorescence detection.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) The stimulated fluorescence is a DC signal, while the background laser is a modulated signal with the same frequency of rotation, which can be effectively distinguished by the filter of the signal processing system;

[0027] (2) Linear polarizers can be installed at any angle because the installation angle only changes the phase of the reflected background laser and does not change the frequency, so it can still be eliminated by filtering.

[0028] (3) Even if the birefringence effect of the sample changes the phase of the reflected background laser, it will not change its frequency and can still be eliminated by filtering. Attached Figure Description

[0029] Figure 1 This is a flowchart of the method of the present invention;

[0030] Figure 2 This is a schematic diagram of the structure of an embodiment of the present invention. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0032] This invention relates to a method for removing background signal interference from excitation fluorescence by rotational polarization modulation. By rotating the incident laser 1, the laser 1 exists in the form of linearly polarized laser until it passes through the linear polarizer 2 to obtain modulated linearly polarized laser. The stimulated fluorescence is obtained by the linearly polarized laser hitting the detection sample 3. After its reflection, it passes through the linear polarizer 2 to obtain DC stimulated fluorescence. Therefore, non-DC light can be filtered out by filtering to obtain the light intensity of stimulated fluorescence, thereby efficiently solving the background interference problem of the light source itself.

[0033] The method includes the following steps:

[0034] The technical solution adopted in this invention is a method for removing background signal interference from excitation fluorescence using rotational polarization modulation, the method comprising the following steps:

[0035] S1 rotates laser 1 at a preset frequency and is incident, and the incident laser 1 is pre-processed;

[0036] In S1, the preprocessing includes filtering the laser 1.

[0037] S2 The pre-processed laser 1 is directed at a preset angle to the dichroic mirror 4 installed at a preset angle, and after being reflected by the dichroic mirror 4, it is emitted towards the test sample 3.

[0038] In S2, the dichroic mirror 4 is set at 45°, with the incident surface of the dichroic mirror 4 facing the incident laser 1 and the detection sample 3.

[0039] In S2, the dichroic mirror 4 reflects the laser 1 into the sample optical detection channel 5. The sample optical detection channel 5 is provided with a first lens 6. A sample position for placing the detection sample 3 is set at the focal length of the first lens 6. After the detection sample 3 is irradiated by the excitation light, it emits fluorescence, which is stimulated fluorescence, and is directed towards the dichroic mirror 4 along the sample optical detection channel 5.

[0040] In this invention, the incident light and reflected light in S1 and S2 are both linearly polarized lasers.

[0041] S3 detects the stimulated fluorescence of sample 3, which is directed toward the dichroic mirror 4. After being transmitted through the dichroic mirror 4, it is modulated with part of the reflected laser 1 to obtain stimulated fluorescence in DC state and modulated polarized laser of the same frequency.

[0042] In S3, the stimulated fluorescence of the detection sample 3 and the reflected laser 1 are modulated by the linear polarizer 2 to obtain stimulated fluorescence in DC state and modulated polarized laser 1 rotating at the same frequency.

[0043] S4 further processes the stimulated fluorescence of S3 and the modulated polarized laser 1 to obtain the output light;

[0044] In S4, the reprocessing includes filtering and focusing the stimulated fluorescence and modulated polarized laser 1 in DC state.

[0045] S5 filters the output light, retaining the stimulated fluorescence information and removing background signal interference.

[0046] In S5, the photosensitive sensor 7 collects the output light signal and filters it to retain the DC signal.

[0047] In this invention, laser 1 enters the sample optical detection channel 5 through reflection by dichroic mirror 4. The excitation source is laser 1, which is linearly polarized light. Laser 1 rotates at a fixed frequency, and its light intensity remains fixed.

[0048] The orientation of the dichroic mirror 4 needs to be determined in advance based on its actual properties, typically 45°; this is something that those skilled in the art can easily understand, and they can set it themselves based on their needs.

[0049] A first lens 6 is set on the optical detection channel 5 of the sample to focus the laser 1 reflected by the dichroic mirror 4 to ensure excitation efficiency; the reflected laser is directly focused onto the sample 3 to be detected. After the sample 3 is excited, it emits fluorescence, and the excited fluorescence returns along the original path and is transmitted through the dichroic mirror 4.

[0050] The partially reflected linearly polarized laser and stimulated fluorescence are modulated in parallel. After the reflected linearly polarized laser passes through linear polarizer 2, the intensity of the light is the cosine component of the angle between the polarization angle of laser 1 and linear polarizer 2. Since laser 1 rotates, a cosine wave with a rotation frequency is generated. The fluorescence signal is only related to the intensity of the excitation source. Before passing through linear polarizer 2, the intensity of the rotating laser 1 is constant, so the intensity of the fluorescence is also constant. Moreover, it is not polarized light. Passing through linear polarizer 2 only results in a fixed proportion of signal loss and does not generate a signal with a certain frequency change. Therefore, through the rotation of laser 1, the light emitted by the excitation source is modulated into a periodic signal with a certain frequency, while the fluorescence signal is a DC component.

[0051] After subsequent filtering and focusing processes, the interference of laser background light can be removed through filtering algorithms.

[0052] Although some energy signal is lost in this invention, the background light is removed more thoroughly, resulting in a fluorescence signal with less interference. The signal-to-noise ratio is greatly improved, and the detection results are more accurate and closer to the self-luminescence sensitivity level of chemiluminescence after using a more sensitive sensor.

[0053] The present invention also relates to a mechanism for removing interference from excitation fluorescence background signal by rotation polarization modulation using the method described above. The mechanism includes a laser incident channel 8 that cooperates with a laser 1, a preprocessing unit provided in cooperation with the laser incident channel 8, and a dichroic mirror 4 provided at the end of the laser incident channel 8 at a preset angle; and a rotation mechanism 9 provided in cooperation with the laser 1.

[0054] A sample optical detection channel 5 is provided on the reflected light side of the dichroic mirror 4, and a sample position for placing the detection sample 3 is provided on the sample optical detection channel 5.

[0055] A fluorescence optical detection channel 10 is provided on the transmission light side of the dichroic mirror 4, and a modulation unit, a reprocessing unit, and a signal processing system are sequentially provided on the fluorescence optical detection channel 10.

[0056] The rotation axis of the rotating mechanism 9 is parallel to the laser incident channel 8, the sample optical detection channel 5 and the fluorescence optical detection channel 10 are coaxial, and the laser incident channel 8 is perpendicular to the sample optical detection channel 5 and the fluorescence optical detection channel 10.

[0057] The dichroic mirror 4 is set at 45°, with its incident surface facing the laser incident channel 8 and the sample optical detection channel 5.

[0058] In this invention, the rotating mechanism 9 is a mechanism that allows the laser 1 to rotate around an axis while keeping its incident light direction unchanged. It keeps the horizontal and vertical positions of the laser 1 unchanged, but allows it to rotate around the incident direction. This is something that those skilled in the art can easily understand, and they can set it up according to their needs.

[0059] In this invention, the preprocessing unit is generally a laser filter 11.

[0060] In this invention, a first lens 6 is provided in the sample optical detection channel 5 to focus the laser 1 onto the sample position.

[0061] In this invention, the modulation unit is a linear polarizer 2, and the reprocessing unit is a fluorescent filter 12 and a second lens 13 arranged in sequence. The second lens 13 focuses the output stimulated fluorescence onto the photosensitive sensor 7.

[0062] In this invention, the signal processing system is equipped with an electronic circuit hardware filter or a software filter to filter out the linearly polarized laser with modulation frequency and retain DC stimulated fluorescence. It is also equipped with a photosensitive sensor 7 to detect light intensity.

[0063] In this embodiment, laser 1, such as a 488nm laser diode, is used as the excitation source and enters the mechanism from the laser incident channel 8. It passes through a pre-processing unit, which is a laser filter 11, such as a 488nm narrowband interference filter 11.

[0064] After the pre-processed laser 1 is directed toward the dichroic mirror 4, it is first focused by the first lens 6 or a lens group to reach the sample position where the test sample 3 is placed. A fluorescence signal is excited at the focal position, such as FAM fluorescent material (fluorescence 525nm). The fluorescence signal is a non-polarized state similar to natural light. The excited fluorescence returns in the opposite direction of the laser 1.

[0065] In addition, some of the laser 1 will also be reflected on the test sample 3 and then passed through the dichroic mirror 4 and transmitted again. That is, some of the laser and stimulated fluorescence can be transmitted through the dichroic mirror 4 and enter the fluorescence optical detection channel 10.

[0066] A modulation unit is set in the fluorescence optical detection channel 10. Part of the reflected laser 1 is modulated to obtain linearly polarized laser with modulation frequency, while the stimulated fluorescence is obtained by passing through the linear polarizer 2 to obtain DC stimulated fluorescence. At the same time, the signal attenuation is close to half.

[0067] The stimulated fluorescence and modulated polarized laser in DC state pass through the post-processing unit, which includes a fluorescence filter 12 and a second lens 13 arranged in sequence. The fluorescence filter 12 can be a narrowband interference filter 12 of 525nm, among others. After the second lens 13 focuses the light, the stimulated fluorescence and modulated polarized laser in DC state enter the photosensitive sensor 7. After the signal is acquired, it is filtered by a filter. The linearly polarized laser of the modulation frequency is filtered out, while the stimulated fluorescence in DC state is retained, thus eliminating the background light interference of the excitation source.

Claims

1. A method for removing background signal interference from excitation fluorescence using rotational polarization modulation, characterized in that: The method includes the following steps: S1 rotates the laser at a preset frequency and is incident, and the incident laser is pre-processed. The pre-processed laser S2 is directed at a preset angle towards a dichroic mirror installed at a preset angle, and after being reflected by the dichroic mirror, it is emitted towards the test sample. The stimulated fluorescence of the S3 detection sample is directed toward the dichroic mirror. After being transmitted through the dichroic mirror, it is modulated with part of the reflected laser to obtain stimulated fluorescence in DC state and modulated polarized laser of the same frequency. S4 further processes the stimulated fluorescence and modulated polarized laser of S3 to obtain the output light; S5 filters the output light, retaining the stimulated fluorescence information and removing background signal interference.

2. The method for removing background signal interference from excitation fluorescence using rotational polarization modulation according to claim 1, characterized in that: In S1, the preprocessing includes laser filtering.

3. The method for removing background signal interference from excitation fluorescence using rotational polarization modulation according to claim 1, characterized in that: In S2, the dichroic mirror is set at 45°, with the incident surface of the dichroic mirror facing the incident laser and the test sample.

4. The method for removing background signal interference from excitation fluorescence using rotational polarization modulation according to claim 3, characterized in that: In S2, the dichroic mirror reflects the laser light into the sample optical detection channel. The sample optical detection channel is provided with a first lens, and a sample position for placing the detection sample is set at the focal length of the first lens. After the detection sample is irradiated by the excitation light, it emits fluorescence, which is stimulated fluorescence, and is directed towards the dichroic mirror along the sample optical detection channel.

5. The method for removing background interference from excitation fluorescence signal using rotational polarization modulation according to claim 3, characterized in that: In S3, a linear polarizer is used to modulate the stimulated fluorescence of the test sample and the reflected laser to obtain stimulated fluorescence in DC state and modulated polarized laser of the same frequency.

6. The method for removing background signal interference from excitation fluorescence using rotational polarization modulation according to claim 1, characterized in that: In S4, the reprocessing includes filtering and focusing the stimulated fluorescence and modulated polarized laser in DC state.

7. The method for removing background interference from excitation fluorescence signal using rotational polarization modulation according to claim 1, characterized in that: In S5, a photosensitive sensor collects the output light signal and filters it to retain the DC signal.

8. A mechanism for removing interference from excitation fluorescence background signal using rotational polarization modulation according to any one of claims 1 to 7, characterized in that: The mechanism includes a laser incident channel that works with the laser, a pre-processing unit that works with the laser incident channel, a dichroic mirror that is positioned at a preset angle at the end of the laser incident channel, and a rotating mechanism that works with the laser. A sample optical detection channel is provided on the reflected light side of the dichroic mirror, and a sample position for placing the detection sample is provided in conjunction with the sample optical detection channel; A fluorescence optical detection channel is provided on the transmission light side of the dichroic mirror, and a modulation unit, a reprocessing unit, and a signal processing system are sequentially provided on the fluorescence optical detection channel.

9. The mechanism for removing background signal interference from excitation fluorescence by rotational polarization modulation according to claim 8, characterized in that: The rotation axis of the rotating mechanism is parallel to the laser incident channel, the sample optical detection channel and the fluorescence optical detection channel are coaxial, and the laser incident channel is perpendicular to the sample optical detection channel and the fluorescence optical detection channel.

10. The mechanism for removing background signal interference from excitation fluorescence using rotational polarization modulation according to claim 8, characterized in that: The dichroic mirror is set at 45°, with its incident surface facing the laser incident channel and the sample optical detection channel.

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