Microscopic circular dichroism spectrum detection system based on single photon counting acquisition method

Abstract

The present application relates to the field of sample optical detection, in particular to a microscopic circular dichroism spectrum detection system based on a single photon counting acquisition method, comprising a light source module, a polarizing module, an excitation module, a microscopic imaging module, a synchronization module, an acquisition module and a data processing module, wherein the light source module generates a wide spectrum continuous light beam, the polarizing module polarizes the wide spectrum continuous light beam into periodically changed left-handed and right-handed polarized light, the periodically changed left-handed and right-handed polarized light interacts in the excitation module, the acquisition module obtains the light intensity signal after the interaction, the data processing module converts the light intensity signal into corresponding circular dichroism spectrum information through mathematical calculation, and the synchronization module provides the timing information for control and acquisition during the whole circular dichroism spectrum acquisition process; the microscopic imaging module can perform microscopic imaging on the measured sample and select the interaction position simultaneously before circular dichroism spectrum acquisition.

Description

Technical Field

[0001] This invention relates to the field of optical sample detection, specifically a microscopic circular dichroism spectroscopy detection system based on a single-photon counting acquisition method. Background Technology

[0002] Circular dichroism spectroscopy is an optical rotation spectrum used to infer the configuration and conformation of asymmetric molecules. Optically active substances have unequal absorption coefficients for left-handed and right-handed polarized light that constitutes plane-polarized light, i.e. Using the spectrum of the plane-polarized light as the abscissa and the difference in absorptivity as the ordinate, The spectrum obtained by plotting the optical active material on the ordinate is the circular dichroism spectrum of the optical active material. The difference in absorption rate can be obtained by measuring the intensity of left-handed and right-handed polarized light transmitted through the optical active material and performing mathematical calculations. Therefore, the main task of the circular dichroism spectroscopy measurement system is to measure the intensity of left-handed and right-handed polarized light transmitted through the optical active material.

[0003] In the currently reported results, modulation methods are used to measure the intensity of left-handed and right-handed polarized light transmitted through the photoactive material under test. This method involves superimposing a periodic modulation signal that matches the period of the left-handed and right-handed polarization of the light intensity signal collected by the photodiode. This method distinguishes the left-handed and right-handed polarized light components of the light intensity signal collected by the photomultiplier tube within one period. This means that in the process of obtaining the left-handed and right-handed polarized light transmitted through the photoactive material under test, the signal must be modulated and demodulated. This process places high demands on the sensitivity and time accuracy of the relevant instruments and will add system noise to the signal. Summary of the Invention

[0004] To address this problem, this invention innovatively employs a single-photon counting method to detect left- and right-handed polarized light transmitted through the photoactive substance being measured. This method can directly convert light intensity into a digital signal, and the corresponding circular dichroism spectrum can be obtained by directly performing digital calculations on the digital signal.

[0005] To meet the requirements for detecting the circular dichroism spectrum of micro-area samples and samples with uneven surface distribution, this invention also includes a microscopic imaging module, enabling the system to meet both micro-area and selected area measurements.

[0006] The purpose of this invention is to provide a microscopic circular dichroism spectral detection system based on a single-photon counting acquisition method. Before measurement, the system utilizes microscopic imaging to achieve microscopic imaging and focusing of the sample, enabling the detection of the circular dichroism of a micro-region or selected area of ​​the sample under test.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] The microscopic circular dichroism spectral detection system based on the single-photon counting acquisition method includes a light source module, a polarization module, an excitation module, and an acquisition module arranged in a straight line. It also includes a data processing module, a microscopic imaging module, a synchronization module, and a white light source.

[0009] The data processing module is connected to the acquisition module and the synchronization module via data transmission lines. The synchronization module is connected to the polarization module via data transmission lines. The microscopic imaging module is located on the refracted light path of the excitation module, and the white light source is located on the reflected light path of the excitation module.

[0010] The light source module includes a broadband continuous light source and two reflectors arranged sequentially on its optical path.

[0011] The polarization module includes a polarizer and a photoelastic modulator arranged in a straight line. The polarizer and the reflector are arranged in a straight line. The synchronization module is connected to the photoelastic modulator through a data transmission line.

[0012] The excitation module includes: a movable beam splitter, a first reflective objective lens, a second reflective objective lens, and a movable reflector arranged in a line. The movable beam splitter and the photoelastic modulator are arranged in a line. The microscopic imaging module is located on the refracted light path of the movable beam splitter. The white light source is located on the reflected light path of the movable reflector. A sample area for placing the sample to be tested is provided between the first reflective objective lens and the second reflective objective lens.

[0013] The acquisition module includes a focusing lens and a grating beam splitter arranged in a straight line, and also includes a photomultiplier tube. The focusing lens and the movable reflector are arranged in a straight line. The photomultiplier tube is located on the reflected light path of the grating beam splitter. The photomultiplier tube is connected to the data processing module through a data transmission line.

[0014] The data processing module includes a single-photon counting acquisition card and a data processing computer. The single-photon counting acquisition card is connected to the synchronization module, the data processing computer, and the photomultiplier tube via data transmission lines.

[0015] The microscopic imaging module includes a focusing mirror and a CCD camera arranged sequentially on the refracting light path of the movable beam splitter, and also includes an imaging display connected to the CCD camera via a data connection line.

[0016] The synchronization module includes a synchronization controller, which is connected to the photoelastic modulator and the single-photon counting acquisition card via data connection lines.

[0017] The microscopic circular dichroism spectroscopy detection method based on single-photon counting includes the following steps:

[0018] When the sample is subjected to spectral measurement, the movable beam splitter and movable reflector in the excitation module are both arranged in a line. The white light emitted by the white light source passes through the movable reflector and the second reflective objective in the excitation module in sequence to illuminate the sample area of ​​the sample under test. The sample area of ​​the sample under test is then imaged in the microscopic imaging module after passing through the first reflective objective and the movable beam splitter in sequence.

[0019] When the spectrum of the sample under test is acquired, the movable beam splitter and movable mirror in the excitation module are removed and arranged in a straight line. The broadband beam emitted by the broadband continuous light source in the light source module passes through the polarization module and the reflective objective in the excitation module in sequence, and is transmitted through the sample area of ​​the sample under test. The transmitted light passes through the second reflective objective and the acquisition module in sequence, and then the spectrum of the sample under test is acquired in the data processing module.

[0020] The present invention has the following beneficial effects and advantages:

[0021] 1. This invention innovatively uses the single-photon counting method to realize the circular dichroism spectral detection of the sample under test, which solves the problem of poor signal-to-noise ratio when using the modulation method for circular dichroism spectral detection in the traditional way, and improves the system acquisition accuracy.

[0022] 2. The present invention is equipped with a microscopic imaging module, which enables the system to have the function of microscopic imaging focusing, thus expanding the technical gap that traditional systems can only perform circular dichroism spectral acquisition on spatially uniform samples. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the system configuration of the present invention;

[0024] Figure 2 This is a schematic diagram of the structure of the present invention;

[0025] Figure 3 This is a schematic diagram illustrating the working principle of the biasing module of the present invention;

[0026] Figure 4 This is a timing control diagram of the present invention;

[0027] Among them, 1 is a broadband continuous light source, 2 is a reflector one, 3 is a reflector two, 4 is a polarizer, 5 is a photoelastic modulator, 6 is a movable beam splitter, 7 is a reflective objective lens one, 8 is the sample under test, 9 is a reflective objective lens two, 10 is a movable reflector, 11 is a focusing lens one, 12 is a grating beam splitter, 13 is a photomultiplier tube, 14 is a white light source, 15 is a focusing lens two, 16 is a CCD camera, 17 is a synchronization controller, 18 is a single-photon counting acquisition card, 19 is a data processing computer, and 20 is an imaging display.

[0028] 501 is a photoelastic modulator controller. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0030] like Figure 1 As shown, the present invention includes a light source module, a polarization module, an excitation module, a microscopic imaging module, a synchronization module, an acquisition module, and a data processing module. Due to the differences in the composition of the modules, the composition of the measurement system differs. Here, one system configuration of the present invention will be further described in detail with reference to the accompanying drawings.

[0031] like Figure 2As shown, this invention includes a light source module, a polarization module, an excitation module, a microscopic imaging module, a synchronization module, an acquisition module, and a data processing module. The light source module includes 1. a broadband continuous light source, 2. a first reflector, and 3. a second reflector. The polarization module includes 4. a polarizer and 5. a photoelastic modulator. The excitation module includes 6. a movable beam splitter, 7. a first reflective objective, 8. the sample under test, 9. a second reflective objective, and 10. a movable reflector. The microscopic imaging module includes 15. a second focusing lens, 16. a CCD camera, and 20. an imaging display. The synchronization module includes a synchronization controller 17. The acquisition module includes 11. a first focusing lens, 12. a grating beam splitter, and 13. a photomultiplier tube. The data processing module includes 18. a single-photon meter. The system includes a data acquisition card, a data processing computer, and a white light source (14). To meet experimental requirements, the system also includes a second reflector (3), a polarizer (4), a photoelastic modulator (5), a movable beam splitter (6), a first reflective objective (7), the sample under test (8), a reflective objective (9), a movable reflector (10), a focusing lens (11), and a grating beam splitter (12) arranged in a straight line. The system's operation includes a microscopic imaging process and a spectral acquisition process. When the movable beam splitter (6) and the movable reflector (10) are connected to the system, the light source module, polarization module, excitation module, microscopic imaging module, and white light source (14) participate in the microscopic imaging process. During microscopic imaging, the white light emitted from the white light source (14) passes sequentially through the movable reflector (10) and the second reflective objective. 9 illuminates the sample area of ​​the test sample 8. The sample area of ​​the test sample 8 passes sequentially through the reflecting objective lens 7 and the movable beam splitter 6 before being imaged in the microscopic imaging module. When the movable beam splitter 6 and the movable mirror 10 are removed from the system, the light source module, polarization module, excitation module, synchronization module, acquisition module, and data processing module participate in the spectral acquisition process. During the spectral acquisition process, the broadband beam emitted from the broadband continuous light source 1 in the light source module changes its propagation direction after being reflected by the mirror 2 and the mirror 3, and then enters the polarization module. The broadband beam is polarized into periodically changing left-handed and right-handed polarized light by the polarizer 4 and the photoelastic modulator 5 in the polarization module before entering the excitation module. At this time, the polarization module moves towards the synchronization control... The trigger signal is provided by the device 17. The synchronization controller 17 provides time delays to the acquisition module and the data processing module according to the specific system conditions, controlling their timing. Since the movable beam splitter 6 and movable reflector 10 in the excitation module are removed from the system, the periodically changing left-handed and right-handed polarized light is focused by the reflective objective lens 7 and transmitted through the sample area of ​​the sample under test 8. The transmitted light is collimated into a parallel beam by the second reflective objective lens 9 and incident on the acquisition module. The parallel beam is focused by the first focusing lens 11 onto the grating beam splitter 12. The grating beam splitter splits the focused beam in the wavelength direction and diffracts it to the photomultiplier tube 13. The photomultiplier tube 13 collects the split single-wavelength beam.The wavelength incident on the photomultiplier tube 13 can be changed by altering the angle of the grating beam splitter 12. Since the excitation light generated by the light source module is polarized by the polarization module into periodically varying left- and right-handed polarized light, the photomultiplier tube 13 also collects periodically varying left- and right-handed polarized light. The photomultiplier tube 13 converts the collected optical signal into an electrical signal and transmits it to the data processing module. The single-photon counting acquisition card 18 in the data processing module controls the timing of the acquisition process, enabling the acquisition module to accurately and completely acquire the left- and right-handed polarized light signals within one period. Therefore, by changing the diffraction wavelength by altering the beam splitter 12, left- and right-handed polarized light signals of different wavelengths can be obtained. The electrical signal converted by the acquisition module is calculated and processed into a circular dichroism spectrum by the data processing computer 19.

[0032] The microscopic imaging process and the spectral acquisition process are switched by a movable beam splitter and a movable mirror.

[0033] The movable beam splitter and the movable reflector have two degrees of freedom: movement and rotation.

[0034] The light source module can consist of a broadband continuous light source, a monochromator, and a light reflecting element, or it can consist of a broadband continuous light source and a light reflecting element.

[0035] The polarization module consists of a linear polarizer and a circular polarizer.

[0036] The linear polarizer can be a linear polarizer or a Glan prism, and the circular polarizer can be a rotatable quarter-wave plate or a photoelastic modulator.

[0037] The excitation module consists of a movable beam splitter, a first reflective objective lens, a sample to be tested, a second reflective objective lens, and a movable mirror. The first and second reflective objective lenses can be replaced with a transmission focusing system.

[0038] The microscopic imaging module consists of a focusing lens, a camera, and an imaging display. In order to achieve the microscopic imaging function, the system is also equipped with a white light source.

[0039] The synchronization module consists of a synchronization controller.

[0040] The acquisition module can be composed of a focusing lens, a grating beam splitter, and a photomultiplier tube, or it can be composed of a focusing lens and a photomultiplier tube. When the light source module is composed of a broadband continuous light source, a monochromator, and a light reflecting element, the acquisition module is composed of a focusing lens and a photomultiplier tube. When the light source module is composed of a broadband continuous light source and a light reflecting element, the acquisition module is composed of a focusing lens, a grating beam splitter, and a photomultiplier tube.

[0041] The data processing module consists of a single-photon counting acquisition card 18 and a data processing computer 19.

[0042] like Figure 3 As shown, the excitation beam emitted from the broadband continuous light source 1 is natural light, that is, the polarization of the direction is the same. When the natural light is incident on the polarization module, the polarizer in the polarization module polarizes the natural light into linearly polarized light and then it is incident on the photoelastic modulator 5. The photoelastic modulator 5 is controlled by the photoelastic modulator controller 501 and exhibits the characteristics of a 1 / 4 wave plate with periodic rotation of the optical axis. Therefore, when the linearly polarized light passes through the photoelastic modulator 5, it becomes periodically changing left-handed and right-handed polarized light.

[0043] like Figure 4 As shown, during the spectral acquisition process, the photoelastic modulator controller 501 generates a square wave signal with a frequency of 50kHz as the trigger signal for the photoelastic modulator 5. When linearly polarized light transmitted through the polarizer 4 is incident on the photoelastic modulator 5, the photoelastic modulator 5 behaves as a quarter-wave plate. The optical axis of the photoelastic modulator 5 rotates periodically under the control of the square wave signal. The optical axis is at a 45° angle to the polarization direction of the linearly polarized light. When the square wave signal is at its rising edge, the optical axis is located in the second and fourth quadrants. When the square wave signal is at its falling edge, the optical axis is located in the first and third quadrants.

[0044] like Figure 4 As shown, during data processing, the 50kHz square wave signal generated by the photoelastic modulator controller 501 triggers the synchronization controller 17. The synchronization controller 17 provides timing control signals to the acquisition module and the data processing module according to the specific system conditions. In order to distinguish the left-handed and right-handed polarized light components corresponding to the acquired light intensity signals, since the absorption rate of the test sample 8 to the left-handed and right-handed polarized light is different, the photomultiplier tube 13 acquires different light intensities during the rotation of the optical axis of the photoelastic modulator 5, thereby obtaining electrical signals with different intensities. The left-handed and right-handed polarized light components corresponding to the electrical signals can be located by synchronizing the timing signals provided by the synchronization controller. After the positions of the electrical signals corresponding to the left-handed and right-handed polarized light are determined, the light intensity information is obtained by intercepting the integral signal within the same gate time.

[0045] The working principle of this invention is as follows:

[0046] like Figure 2As shown, the excitation module of this invention includes a movable beam splitter 6 and a movable mirror 10. During microscopic imaging of the sample 8, the movable beam splitter 6 moves between the photoelastic modulator 5 and the first reflecting objective lens 7, and the movable mirror 10 moves between the second reflecting objective lens 9 and the first focusing lens 11. During the microscopic imaging operation, the excitation beam emitted from the broadband continuous light source 1 in the light source module passes sequentially through the first reflecting mirror 2, the second reflecting mirror 3, the polarizer 4, the photoelastic modulator 5, the movable beam splitter 6, and the second reflecting objective lens 7 before being focused onto the sample area of ​​the sample 8. The focal point of the excitation beam coincides with the sample area. The white light emitted from the white light source 14 passes sequentially through the movable reflecting mirror 10 and the second reflecting objective lens 9 before illuminating the sample. The sample area of ​​sample 8, along with the focal point of the excitation beam, passes sequentially through the reflecting objective lens 7 and the movable beam splitter 6 before being reflected into the microscopic imaging module. Sample 8 is fixed on a translation stage with XYZ degrees of freedom. By adjusting the translation stage, the position of sample 8 is changed, so that the sample area of ​​sample 8 and the focal point of the excitation beam form a clear and complete image in the microscopic imaging module. The imaging display can display the clear and complete image. By adjusting the reflecting mirror 2 and the reflecting mirror 3, the overlap position between the focal point of the excitation beam and the sample area is changed. This operation changes the excitation position of the sample area, making it possible to select and detect the sample 8 in the subsequent spectral measurement process.

[0047] like Figure 2 As shown, the excitation module of this invention includes a movable beam splitter 6 and a movable reflector 10. When performing spectral measurements on the sample 8, the movable beam splitter 6 and the movable reflector 10 in the excitation module are moved out of the system. After imaging and focusing in the microscopic imaging process, the excitation position of the sample area of ​​the sample 8 has been selected. At this time, the beam emitted from the broadband continuous light source 1 is reflected by reflector 2 and reflector 3 to the polarizer 4. The beam that passes through the polarizer 4 and the photoelastic modulator 5 in sequence is polarized into periodically changing left-handed and right-handed polarized light. The periodically changing left-handed polarized light... Right-handed polarized light is focused onto the sample area of ​​the test sample 8 by a reflective objective lens and transmitted through the test sample 8. The transmitted light that has passed through the test sample 8 is collimated into a parallel beam by a second reflective objective lens 9 and then focused onto a grating beam splitter 12 by a first focusing lens 11. The grating beam splitter 12 splits the focused beam in the wavelength direction. The grating beam splitter 12 is fixed on a displacement stage with a degree of freedom of rotation around the Z-axis. The displacement stage can drive the grating beam splitter 12 to rotate around the Z-axis under the drive of a motor. By rotating the grating beam splitter around the Z-axis, the wavelength received by the photomultiplier tube 13 can be changed.

[0048] like Figure 2As shown, during data acquisition and processing, the photoelastic modulation controller 501 generates a square wave signal with a period of 50kHz. The square wave signal controls the rotation of the optical axis of the photoelastic modulator 5, causing the excitation light transmitted through the photoelastic modulator to exhibit periodically changing left-handed and right-handed polarized light. The square wave signal with a period of 50kHz simultaneously triggers the synchronization controller 17. The synchronization controller provides timing signals to the photomultiplier tube and the single-photon counting acquisition card 18 according to the actual system situation. The single-photon counting acquisition card 18 calibrates the acquisition timing of the photomultiplier tube 13 and distinguishes the left-handed and right-handed polarized light components of the light signal acquired by the photomultiplier tube 13. During data processing, the intensity of left-handed and right-handed polarized light of a single wavelength can be obtained through a single acquisition. By rotating the grating beam splitter 12 to change the wavelength, the circular dichroism spectrum of the test sample 8 in the full spectrum can be obtained.

[0049] In this embodiment, the broadband continuous light source 1 is preferably a deuterium-halogen lamp, and the first reflective objective lens 7 and the second reflective objective lens 9 are preferably 200mm focal length objectives.

Claims

1. A microscopic circular dichroism spectral detection system based on a single-photon counting acquisition method, characterized in that, It includes a light source module, a polarization module, an excitation module and a data acquisition module arranged in a line, as well as a data processing module, a microscopic imaging module, a synchronization module and a white light source (14). The data processing module is connected to the acquisition module and the synchronization module respectively via a data transmission line. The synchronization module is connected to the polarization module via a data transmission line. The microscopic imaging module is located on the refracted light path of the excitation module. The white light source (14) is located on the reflected light path of the excitation module. The light source module includes a broadband continuous light source (1) and a reflector one (2) and a reflector two (3) arranged sequentially on its optical path. The polarization module includes a polarizer (4) and a photoelastic modulator (5) arranged in a straight line. The polarizer (4) and the second reflector (3) are arranged in a straight line. The synchronization module is connected to the photoelastic modulator (5) through a data transmission line. The acquisition module includes a focusing lens (11) and a grating beam splitter (12) arranged in a line, and also includes a photomultiplier tube (13). The focusing lens (11) and the movable reflector (10) are arranged in a line. The photomultiplier tube (13) is located on the reflected light path of the grating beam splitter (12). The photomultiplier tube (13) is connected to the data processing module through a data transmission line.

2. The microscopic circular dichroism spectral detection system based on the single-photon counting acquisition method according to claim 1, characterized in that, The excitation module includes: a movable beam splitter (6), a first reflective objective lens (7), a second reflective objective lens (9), and a movable reflector (10) arranged in a line. The movable beam splitter (6) and the photoelastic modulator (5) are arranged in a line. The microscopic imaging module is located on the refracted light path of the movable beam splitter (6). The white light source (14) is located on the reflected light path of the movable reflector (10). A sample area for placing the sample to be tested (8) is provided between the first reflective objective lens (7) and the second reflective objective lens (9).

3. The microscopic circular dichroism spectral detection system based on the single-photon counting acquisition method according to claim 1, characterized in that, The data processing module includes a single-photon counting acquisition card (18) and a data processing computer (19). The single-photon counting acquisition card (18) is connected to the synchronization module, the data processing computer (19) and the photomultiplier tube (13) respectively via a data transmission line.

4. The microscopic circular dichroism spectral detection system based on the single-photon counting acquisition method according to claim 1, characterized in that, The microscopic imaging module includes a focusing lens 2 (15) and a CCD camera (16) arranged sequentially on the refracting optical path of the movable beam splitter (6), and also includes an imaging display (20) connected to the CCD camera (16) via a data connection line.

5. The microscopic circular dichroism spectral detection system based on the single-photon counting acquisition method according to claim 1, characterized in that, The synchronization module includes a synchronization controller (17), which is connected to the photoelastic modulator (5) and the single-photon counting acquisition card (18) via data connection lines.

6. A microscopic circular dichroism spectral detection method based on single-photon counting acquisition, applied to the microscopic circular dichroism spectral detection system based on single-photon counting acquisition as described in claim 2, characterized in that, Includes the following steps: When the sample under test is subjected to spectral measurement, the movable beam splitter (6) and the movable mirror (10) in the excitation module are both arranged in a line. The white light emitted by the white light source (14) passes through the movable mirror (10) and the second reflective objective lens (9) in the excitation module to illuminate the sample area of ​​the sample under test (8). The sample area of ​​the sample under test (8) passes through the first reflective objective lens (7) and the movable beam splitter (6) in sequence and is then imaged in the microscopic imaging module. When the spectrum of the sample under test is acquired, the movable beam splitter (6) and the movable mirror (10) in the excitation module are removed and arranged in a straight line. The broadband beam emitted by the broadband continuous light source (1) in the light source module passes through the polarization module and the first reflective objective (7) in the excitation module in sequence, and is transmitted through the sample area of ​​the sample under test (8). The transmitted light passes through the second reflective objective (9) and the acquisition module in sequence, and then the spectrum of the sample under test (8) is acquired in the data processing module.

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