Single-molecule fluorescence spectrum imaging device and calibration method thereof

Abstract

The invention discloses a single-molecule fluorescence spectrum imaging device and a calibration method thereof, the imaging device comprises a diaphragm, a first lens, a first reflector module, a dispersion prism, a second reflector module, a second lens and a camera, the first reflector module and the second reflector module can be moved in or out of a light path at the same time; if the first reflector module and the second reflector module move into the light path at the same time, incident light sequentially passes through the diaphragm, the first lens, the first reflector module, the dispersion prism, the second reflector module and the second lens and then is imaged in the camera; if the first reflector module and the second reflector module move out of the light path at the same time, the incident light sequentially passes through the diaphragm, the first lens and the second lens and then is imaged in the camera. The embodiment of the invention can improve the measurement precision and reduce the cost in the full view field range, and can be widely applied to the technical field of detection.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a single-molecule fluorescence spectrum imaging device and a calibration method thereof. Background Art

[0002] Single-molecule fluorescence technology has been applied to a variety of studies, such as tracking the movement of motor proteins, analyzing the spatial distribution and organization of nucleic acids and proteins in cells, and monitoring conformational changes of biomolecules and exploring their physiological functions. When single-molecule fluorescence technology was invented, it was originally used as a spectral analysis tool, but now it is more often measured using fluorescence intensity rather than fluorescence spectroscopy. However, single-molecule fluorescence spectroscopy has once again attracted attention in recent studies because the molecular spectral features it contains have many potential application scenarios, including low-crosstalk multicolor fluorescence detection for super-resolution imaging and DNA sequencing, chemical reaction path analysis, and polarity and viscosity monitoring of cellular microenvironments. Therefore, it is crucial to develop a simple and easy-to-use spectral single-molecule fluorescence detection device.

[0003] Existing single-molecule fluorescence spectral imaging devices usually require two optical paths to record images in imaging mode and spectral mode respectively. To this end, dual objective lenses and dual cameras are needed, resulting in high costs; or the field of view of a camera is divided into two halves, resulting in a smaller field of view.

[0004] In addition, traditional single-molecule fluorescence spectroscopy measurement devices usually integrate dispersive components, such as gratings or prisms. Adding gratings or prisms to the single-molecule fluorescence detection optical path will introduce image distortion, resulting in differences in the wavelength-pixel relationship at different positions in the field of view. Therefore, when measuring the spectra of multiple fluorescent molecules simultaneously within a large field of view, the measurement accuracy will be significantly reduced. In addition, in order to calibrate the wavelength-pixel relationship, traditional methods usually require the use of lasers with multiple wavelengths as monochromatic light sources, which not only increases costs but also brings the risk of damaging the camera. Summary of the invention

[0005] In view of this, in order to solve one of the above problems, an embodiment of the present invention aims to provide a single-molecule fluorescence spectroscopy imaging device and a calibration method thereof, which can improve measurement accuracy and reduce costs within the entire field of view.

[0006] On the one hand, an embodiment of the present invention provides a single-molecule fluorescence spectral imaging device, including an aperture, a first lens, a first reflector module, a dispersion prism, a second reflector module, a second lens and a camera, wherein the first reflector module and the second reflector module can be moved into or out of the optical path at the same time; if the first reflector module and the second reflector module are moved into the optical path at the same time, the incident light passes through the aperture, the first lens, the first reflector module, the dispersion prism, the second reflector module and the second lens in sequence, and then is imaged on the camera; if the first reflector module and the second reflector module are moved out of the optical path at the same time, the incident light passes through the aperture, the first lens and the second lens in sequence, and then is imaged on the camera.

[0007] Optionally, the aperture comprises a two-dimensionally adjustable aperture.

[0008] Optionally, the aperture and the camera satisfy a conjugate relationship.

[0009] Optionally, the imaging device further comprises a light source, a filter block and an objective lens, and the light emitted by the light source sequentially passes through the filter block and the objective lens to reach the sample to be tested, so that the sample to be tested emits fluorescence, and the fluorescence passes through the filter block to form incident light.

[0010] The implementation of the embodiment of the present invention includes the following beneficial effects: the single-molecule fluorescence spectral imaging device includes an aperture, a first lens, a first reflector module, a dispersion prism, a second reflector module, a second lens and a camera, and the first reflector module and the second reflector module can be moved into or out of the optical path at the same time; if the first reflector module and the second reflector module are moved into the optical path at the same time, the incident light passes through the aperture, the first lens, the first reflector module, the dispersion prism, the second reflector module and the second lens in sequence, and then forms an image on the camera; if the first reflector module and the second reflector module are moved out of the optical path at the same time, the incident light passes through the aperture, the first lens and the second lens in sequence, and then forms an image on the camera, and the first reflector module and the second reflector module are moved into or out of the optical path to switch between the imaging mode and the spectral mode, without using dual objective lenses or dual cameras, thereby reducing costs, and at the same time, the imaging mode and the spectral mode both include a full field of view range, thereby realizing a single-molecule spectral measurement with a full field of view range and high throughput of a single camera, thereby improving measurement accuracy.

[0011] On the other hand, an embodiment of the present invention provides a calibration method for a single-molecule fluorescence spectrum imaging device, which is applied to the above imaging device, and includes:

[0012] Simultaneously moving the first reflector module and the second reflector module out of the light path, acquiring a first image of the microsphere, and determining a first spatial position coordinate of the microsphere according to the first image;

[0013] Simultaneously moving the first reflector module and the second reflector module into the optical path, acquiring a first spectral band of the microsphere, and determining a first reference coordinate according to the first spectral band;

[0014] A first calibration coefficient is determined based on the first spatial position coordinates and the first reference coordinates.

[0015] The implementation of the embodiment of the present invention includes the following beneficial effects: during the calibration process of the single-molecule fluorescence spectral imaging device, first, the first reflector module and the second reflector module are simultaneously moved out of the optical path to obtain a first image of the microsphere, and the first spatial position coordinates of the microsphere are determined based on the first image, that is, the coordinates in the imaging mode are obtained; then, the first reflector module and the second reflector module are simultaneously moved into the optical path to obtain a first spectral band of the microsphere, and the first reference coordinates are determined based on the first spectral band, that is, the coordinates in the spectral mode are obtained; finally, the first calibration coefficient is determined based on the first spatial position coordinates and the first reference coordinates, so that image distortion can be corrected, spectral data collection within the entire field of view is achieved, image distortion can be corrected, and the spectral measurement results within the entire field of view have high uniformity, thereby improving the spectral measurement accuracy.

[0016] Optionally, the method further comprises:

[0017] Simultaneously moving the first reflector module and the second reflector module out of the light path, acquiring a second image of the microsphere, and determining a second spatial position coordinate of the microsphere according to the second image;

[0018] Simultaneously moving the first reflector module and the second reflector module into the optical path, acquiring a second spectral band of the microsphere, and determining the center coordinates of a light intensity peak or a light intensity valley according to the second spectral band;

[0019] Determine a second reference coordinate according to the second spatial position coordinate and the first calibration coefficient, and determine a pixel offset according to the center coordinate and the second reference coordinate;

[0020] The second calibration coefficient is determined according to the central wavelength of the light intensity peak or the light intensity valley and the pixel offset.

[0021] The implementation of the embodiment of the present invention includes the following beneficial effects: using the fluorescence emitted by the microsphere as the incident light, combined with a multi-channel filter block, to determine the pixel offset according to the center coordinates of the light intensity peak or the light intensity valley according to the second spectral band and the calibrated second reference coordinates, determining the second calibration coefficient according to the center wavelength of the light intensity peak or the light intensity valley in the second spectral band and the pixel offset, accurately mapping the pixel to the wavelength, and not needing to use multiple lasers as a reference for wavelength-pixel relationship calibration, lowering the cost and minimizing the risk of camera damage.

[0022] Optionally, determining a first calibration coefficient according to the first spatial position coordinates and the first reference coordinates includes:

[0023] Taking the first reference coordinate as a first dependent variable and the first spatial position coordinate as a first independent variable;

[0024] A first calibration coefficient is determined by performing fitting according to the first dependent variable, the first independent variable and a preset first polynomial.

[0025] Optionally, determining the second calibration coefficient according to the central wavelength of the light intensity peak or the light intensity valley and the pixel offset includes:

[0026] Taking the central wavelength of the light intensity peak or light intensity valley as a second dependent variable, and taking the pixel offset as a second independent variable;

[0027] A second calibration coefficient is determined by performing fitting according to the second dependent variable, the second independent variable and a preset second polynomial.

[0028] Optionally, determining the pixel offset according to the center coordinate and the second reference coordinate includes:

[0029] respectively calculating a plurality of differences between the central coordinates of a plurality of light intensity peaks or light intensity valleys and the second reference coordinates;

[0030] An average value of several of the difference values ​​is determined as the pixel shift.

[0031] On the other hand, an embodiment of the present invention provides a calibration device for a single-molecule fluorescence spectroscopy imaging device, comprising:

[0032] at least one processor;

[0033] at least one memory for storing at least one program;

[0034] When the at least one program is executed by the at least one processor, the at least one processor implements the above method.

[0035] The implementation of the embodiment of the present invention includes the following beneficial effects: the calibration device and the calibration method of the single-molecule fluorescence spectrum imaging device achieve the same effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a light path diagram of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0037] Figure 2 is a light path diagram of another single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0038] Figure 3 is a light path diagram of another single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0039] Figure 4 It is a schematic diagram of the steps of a calibration method of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0040] Figure 5 is a result diagram of a calibration method for a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0041] Figure 6 It is a schematic flow chart of the steps of another calibration method of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0042] Figure 7 is a result diagram of another calibration method of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0043] Figure 8 It is a schematic flow chart of the steps of another calibration method of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0044] Fig. 9 is a result diagram of another calibration method of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0045] Fig.10 is a result diagram of another calibration method of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0046] Fig.11 is a result diagram of another calibration method of a single-molecule fluorescence spectrum imaging device provided by an embodiment of the present invention;

[0047] Fig.12 It is a structural block diagram of a calibration device for a single-molecule fluorescence spectral imaging device provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The step numbers in the following embodiments are only provided for the convenience of explanation and description, and the order between the steps is not limited in any way. The execution order of each step in the embodiment can be adaptively adjusted according to the understanding of those skilled in the art.

[0049] Some technical terms are explained below.

[0050] Filter: An optical component that selectively transmits or reflects certain wavelengths of light.

[0051] Passband: refers to the wavelength range of light that the filter allows to pass through. This part of the light can transmit the filter.

[0052] Cut-off band: refers to the wavelength range of light that the filter does not allow to pass through. This part of the light is reflected and cannot pass through the filter.

[0053] Filter block: It can be regarded as a combination of multiple filters, usually composed of three parts: excitation filter, emission filter and dichroic mirror. It is the core component used for fluorescence imaging in fluorescence microscope and is installed in the filter wheel of fluorescence microscope.

[0054] Fluorescent molecules: molecules that absorb light energy and then emit light. Usually, the wavelength of the emitted light is longer than the wavelength of the absorbed light. For example, when a 532-nanometer laser is used to irradiate a Cy5 molecule, it can emit fluorescence. The curve of the distribution of fluorescence intensity over wavelength is called the fluorescence spectrum. The position with the highest intensity is the peak position, and the corresponding wavelength is the peak wavelength. For example, the peak wavelength of the fluorescence spectrum of the Cy5 molecule is 680 nanometers.

[0055] Single-step bleaching: When fluorescent molecules are exposed to light for a long time, they will lose their ability to emit light, which is called bleaching. If the intensity of fluorescence is observed to be gradually weakened, it means that multiple fluorescent molecules are gathered together and each molecule is bleached in turn. If fluorescence bleaching is observed to drop from "yes" to "no" at one time (i.e. single-step bleaching), it means that there is only one fluorescent molecule here (i.e. single molecule).

[0056] like Figure 1 As shown, an embodiment of the present invention provides a single-molecule fluorescence spectrum imaging device, comprising an aperture (1), a first lens (2), a first reflector module, a dispersion prism (5), a second reflector module, a second lens (8) and a camera (9), wherein the first reflector module and the second reflector module can be moved into or out of the optical path at the same time; if the first reflector module and the second reflector module are moved into the optical path at the same time, the incident light passes through the aperture (1), the first lens (2), the first reflector module, the dispersion prism (5), the second reflector module and the second lens (8) in sequence, and then forms an image on the camera (9), which is referred to as a "spectral mode" in this embodiment; see Figure 2 If the first reflector module and the second reflector module are simultaneously moved out of the optical path, the incident light passes through the aperture (1), the first lens (2) and the second lens (8) in sequence, and is imaged by the camera (9), which is referred to as the "imaging mode" in this embodiment.

[0057] It will be understood by those skilled in the art that, in the imaging mode, the data recorded by the camera (9) is the image of a single-molecule fluorescence point; in the spectral mode, the point light source on the aperture (1) is broadened into a spectral strip by the dispersion prism (5), and at this time, the data recorded by the camera (9) is the spectral image of the single-molecule fluorescence.

[0058] It should be noted that the first reflector module is used to transmit the output light of the first lens (2) to the dispersion prism (5), and the second reflector module is used to transmit the output light of the dispersion prism (5) to the second lens (8). The first reflector module and the second reflector module are determined according to actual applications and are not specifically limited in this embodiment. Figure 1 In a specific embodiment, the first reflector module includes a first movable reflector (3) and a first reflector (4), and the second reflector module includes a second reflector (6) and a second movable reflector (7). The first movable reflector (3) and the second movable reflector (7) can be mounted on an electric rotating frame, and both can be moved into or out of the optical path at the same time.

[0059] The incident light passes through the object plane aperture (1) and reaches the first lens (2). The first lens (2) transforms the light emitted by the point light source on the object plane into parallel light. The first movable reflector (3) is used to reflect the parallel light emitted by the first lens (2) to the first reflector (4). The first reflector (4) is used to guide the light from the first movable reflector (3) to the dispersion prism (5). The dispersion prism (5) is used to deflect light of different wavelengths to different angles. The second reflector (6) is used to guide the light emitted from the dispersion prism (5) to the second movable reflector (7). The second movable reflector (7) is used to reflect the parallel light emitted by the second reflector (6) to the second lens (8). The second lens (8) re-converges the parallel light into an imaging point, and forms an image on the camera (9).

[0060] It should be noted that the dispersion prism (5) is determined according to actual application, and this embodiment does not impose any specific restrictions. For example, the dispersion prism (5) is an equilateral prism made of calcium fluoride (CaF2). The structure and focal length and other parameters of the first lens (2) and the second lens (8) are determined according to actual application, and this embodiment does not impose any specific restrictions. For example, the first lens (2) and the second lens (8) are both double-cemented achromatic lenses with a focal length of 100 mm.

[0061] The camera (9) is determined according to actual application, and this embodiment does not make specific restrictions. For example, the camera (9) is a two-dimensional array photodetector, including but not limited to an electron-multiplying charge-coupled device (EMCCD) and a scientific complementary metal-oxide-semiconductor (sCMOS). The camera (9) is used to collect image and video data.

[0062] Optionally, the aperture comprises a two-dimensionally adjustable aperture.

[0063] It should be noted that the specific structure of the diaphragm is determined according to the actual application, and this embodiment does not impose any specific restrictions. In a specific embodiment, the diaphragm is a two-dimensional adjustable rectangular hole for adjusting the size of the field of view. Further, the diaphragm is adjusted so that the size of the field of view is consistent with the sensor size of the camera, thereby improving the measurement accuracy. It can be understood by those skilled in the art that there is a microscope in front of the diaphragm, and the plane where the diaphragm is located is the imaging surface of the microscope.

[0064] Optionally, the aperture and the camera satisfy a conjugate relationship.

[0065] Through conjugate imaging, the distortion and error in the imaging process can be effectively eliminated or reduced, thereby improving the quality and accuracy of imaging.

[0066] Optionally, see Figure 3 The imaging device also includes a light source, a filter block (1-3) and an objective lens (1-2). The light emitted by the light source sequentially passes through the filter block (1-3) and the objective lens (1-2) to reach the sample (1-1) to be tested, so that the sample (1-1) to be tested emits fluorescence. The fluorescence passes through the filter block (1-3) to form incident light and is incident on the device (1-4). The device (1-4) includes an aperture, a first lens, a first reflector module, a dispersion prism, a second reflector module, a second lens and a camera.

[0067] For the sake of clarity, the X direction and Y direction of the image captured by the camera (9) are defined as follows: Figure 3 As shown, the X direction is perpendicular to the bottom surface of the prism and points inward, and the Y direction is within the bottom surface of the prism and points from the bottom edge of the prism to the direction of the prism vertex.

[0068] It should be noted that the number of channels of the filter block is determined according to actual applications, and this embodiment does not impose any specific limitation, such as a dual-channel filter block or a four-channel filter block.

[0069] The implementation of the embodiment of the present invention includes the following beneficial effects: the single-molecule fluorescence spectral imaging device includes an aperture, a first lens, a first reflector module, a dispersion prism, a second reflector module, a second lens and a camera, and the first reflector module and the second reflector module can be moved into or out of the optical path at the same time; if the first reflector module and the second reflector module are moved into the optical path at the same time, the incident light passes through the aperture, the first lens, the first reflector module, the dispersion prism, the second reflector module and the second lens in sequence, and then forms an image on the camera; if the first reflector module and the second reflector module are moved out of the optical path at the same time, the incident light passes through the aperture, the first lens and the second lens in sequence, and then forms an image on the camera, and the first reflector module and the second reflector module are moved into or out of the optical path to switch between the imaging mode and the spectral mode, without using dual objective lenses or dual cameras, thereby reducing costs, and at the same time, the imaging mode and the spectral mode both include a full field of view range, thereby realizing a single-molecule spectral measurement with a full field of view range and high throughput of a single camera, thereby improving measurement accuracy.

[0070] When the device switches from imaging mode to spectroscopy mode, a prism is introduced into the optical path, which causes additional image distortion. In order to correct the image distortion to achieve accurate spectral measurement over the full field of view, the image distortion needs to be characterized, that is, the spatial position coordinates of the imaging mode are related to the reference coordinates of the spectroscopy mode.

[0071] See also Figure 4 The embodiment of the present invention provides a calibration method for a single-molecule fluorescence spectrum imaging device, which is applied to the above-mentioned imaging device, and includes:

[0072] S100, simultaneously moving the first reflector module and the second reflector module out of the light path, acquiring a first image of the microsphere, and determining a first spatial position coordinate of the microsphere according to the first image;

[0073] S200, moving the first reflector module and the second reflector module into the optical path at the same time, acquiring a first spectral band of the microsphere, and determining a first reference coordinate according to the first spectral band;

[0074] S300, determining a first calibration coefficient according to a first spatial position coordinate and a first reference coordinate.

[0075] It should be noted that, in this embodiment, the sample to be tested is a microsphere, and the microsphere is a broad-spectrum fluorescent microsphere. In a specific embodiment, when irradiated with a short-wave laser, the fluorescence emitted by the microsphere covers a wavelength range of 450 nanometers to 750 nanometers.

[0076] It should be noted that the reference coordinates of the spectral strip refer to the coordinates of the light intensity extreme value points on the spectral strip, including but not limited to the coordinates of the light intensity maximum value point and the light intensity minimum value point.

[0077] Specifically, the first reflector module and the second reflector module are simultaneously moved out of the optical path, that is, a first image of the microsphere is collected in the imaging mode, and a first spatial position coordinate (x im1 ,y im1 ); move the first reflector module and the second reflector module into the optical path at the same time, that is, collect the first spectral band of the microsphere in the spectral mode, and calculate the first reference coordinate (x sp1 ,y sp1 ); with the first spatial position coordinate (x im1 ,y im1 ) is the independent variable, the first reference coordinate (x sp1 ,y sp1 ) is the dependent variable, and curve fitting is performed to determine the first calibration coefficient.

[0078] Optionally, determining a first calibration coefficient according to the first spatial position coordinate and the first reference coordinate includes:

[0079] S310, taking the first reference coordinate as a first dependent variable, and taking the first spatial position coordinate as a first independent variable;

[0080] S320, performing fitting according to the first dependent variable, the first independent variable and a preset first polynomial to determine a first calibration coefficient.

[0081] It should be noted that the specific form of the first polynomial is determined according to actual applications and is not specifically limited in this embodiment.

[0082] In a specific embodiment, fluorescent microspheres are immobilized on the surface of a glass slide and characterized using a dual-channel filter cube.

[0083] See also Figure 5 , the calibration method of image distortion is as follows:

[0084] Step S11: photographing the first image of the fluorescent microspheres in the imaging mode and the first spectral band in the spectral mode respectively.

[0085] Step S12: For the first image in the imaging mode, mark the position of the fluorescent microspheres.

[0086] Step S13: Perform two-dimensional Gaussian fitting on the data in the 20×20 pixel area around the marked position to obtain more accurate spatial position coordinates (x im1 ,y im2 ),like Figure 5 As shown in (a).

[0087] Step S14: For the first spectral band of the spectral pattern, extract the coordinates (x im1 ,y im1 ) is used to calculate the first reference coordinate (x sp1 ,y sp1 Specifically, for the X direction, the data in the region are added along the Y axis to obtain the X profile, and the x sp1 Similarly, for the Y direction, the data in the region are added along the X axis to obtain the Y profile. Since there is a cutoff band between the two passbands of the dual-channel filter block, there is a peak and valley in the Y profile. Gaussian fitting is performed on the data in the peak and valley to obtain y sp1 . Figure 5 (b) is the first spectral band of the fluorescent microspheres in the spectral mode. The gap in the first spectral band is caused by the cutoff band of the filter block, and its center is used as the first reference coordinate (x sp1 ,y sp1 ), Figure 5 The data in the rectangular box in (b) is taken out and used to calculate the first reference coordinates. Figure 5 (c) shows the calculation of the x component of the first reference coordinate (x sp1 ) method: First Figure 5 The data in the rectangular box of (b) are summed along the Y axis to obtain the light intensity profile data along the X direction ( Figure 5 The light intensity profile data is fitted with a Gaussian function to obtain the x component of the reference coordinate. Figure 5 (d) shows the calculation of the y component of the first reference coordinate (y sp1 ) method: First Figure 5 The data in the rectangular box in (b) are summed along the X axis to obtain the light intensity profile data along the Y direction ( Figure 5 The data in the peaks and valleys are fitted with a Gaussian function to obtain the y component of the first reference coordinate.

[0088] Step S15: Repeat steps S11-S15 to calculate the first spatial position coordinates (x im1 ,y im1 ) set and the first reference coordinate (x sp1 ,y sp1 )gather.

[0089] Step S16: Use a binary second-order polynomial (Formula (1)) to fit the first spatial position coordinate (x im1 ,y im1 ) set and the first reference coordinate (x sp1 ,y sp1 ) set, the obtained polynomial coefficients are the result of the image distortion characterization. Specifically, in the fitting process, the least square method can be used to determine the polynomial coefficients.

[0090]

[0091] Among them, a 20 、a 02 、a 11 、a 10 、a 01 、a 00 、b 20 、b 02 、b 11 、b 10 、b 01 、b 00 These are all coefficients to be determined.

[0092] The implementation of the embodiment of the present invention includes the following beneficial effects: during the calibration process of the single-molecule fluorescence spectral imaging device, first, the first reflector module and the second reflector module are simultaneously moved out of the optical path to obtain a first image of the microsphere, and the first spatial position coordinates of the microsphere are determined based on the first image, that is, the coordinates in the imaging mode are obtained; then, the first reflector module and the second reflector module are simultaneously moved into the optical path to obtain a first spectral band of the microsphere, and the first reference coordinates are determined based on the first spectral band, that is, the coordinates in the spectral mode are obtained; finally, the first calibration coefficient is determined based on the first spatial position coordinates and the first reference coordinates, so that image distortion can be corrected, spectral data collection within the entire field of view is achieved, image distortion can be corrected, and the spectral measurement results within the entire field of view have high uniformity, thereby improving the spectral measurement accuracy.

[0093] In spectral mode, the fluorescent image point is broadened into a spectral strip. The spectral data captured by the camera is summed along the X direction to obtain the light intensity profile curve along the Y direction, with the ordinate being the light intensity and the abscissa being the pixel. The ordinate of the spectrum is the light intensity and the abscissa is the wavelength. In order to convert the light intensity profile curve into a spectrum, that is, to convert the abscissa from pixels to wavelength, the wavelength-pixel relationship needs to be calibrated in advance.

[0094] Optionally, see Figure 6 , the calibration method of the single-molecule fluorescence spectroscopy imaging device also includes:

[0095] S400, simultaneously moving the first reflector module and the second reflector module out of the light path, acquiring a second image of the microsphere, and determining a second spatial position coordinate of the microsphere according to the second image;

[0096] S500, moving the first reflector module and the second reflector module into the optical path at the same time, acquiring a second spectral band of the microsphere, and determining the center coordinates of the light intensity peak or the light intensity valley according to the second spectral band;

[0097] S600, determining a second reference coordinate according to the second spatial position coordinate and the first calibration coefficient, and determining a pixel offset according to the center coordinate and the second reference coordinate;

[0098] S700, determining a second calibration coefficient according to the central wavelength of the light intensity peak or the light intensity valley and the pixel offset.

[0099] It should be noted that the light intensity peak or light intensity valley is caused by the multi-channel filter block. The light intensity peak corresponds to the passband of the filter block, and the position of the light intensity peak corresponds to the center wavelength position of the passband. The light intensity valley corresponds to the cut-off band of the filter block, and the position of the light intensity valley value corresponds to the center wavelength position of the cut-off band. Referring to the filter block transmittance curve, the center wavelength (λ) of the passband and cut-off band can be calculated.

[0100] Specifically, the first reflector module and the second reflector module are simultaneously moved into the optical path, that is, the second image of the microsphere is collected in the imaging mode, and the second spatial position coordinate (x im2 ,y im2 ); using the image distortion characterization result, i.e., the first calibration coefficient, the second spatial position coordinate (x im2 ,y im2 ) is converted to the second reference coordinate (x sp2 ,y sp2 ); collect the second spectral band of the microsphere in the spectral mode, and calculate the center coordinates (y s ′ p ), calculate the center coordinates (y s ′ p ) relative to the second reference coordinate (y sp2 ) pixel offset (Δy sp ); calculate the central wavelength (λ) corresponding to the peak or valley of light intensity, and use the pixel offset (Δy sp ) is taken as the independent variable and the wavelength (λ) is taken as the dependent variable to perform curve fitting. The fitting parameters obtained are the results of spectrum calibration, i.e., the second calibration coefficients.

[0101] Optionally, determining the pixel offset according to the center coordinate and the second reference coordinate includes:

[0102] S610, respectively calculating a plurality of differences between the central coordinates of a plurality of light intensity peaks or light intensity valleys and a second reference coordinate;

[0103] S620: Determine an average value of the plurality of difference values ​​as a pixel offset.

[0104] It should be noted that the number of differences is determined according to actual applications, and this embodiment does not impose any specific restrictions. A difference is calculated based on the center coordinates of a light intensity peak and the second reference coordinates, and / or a difference is calculated based on the center coordinates of a light intensity valley and the second reference coordinates, and then the pixel offset is determined based on the average value of several differences. The pixel offset is determined based on the average value of multiple differences, which can reduce errors.

[0105] Optionally, determining a second calibration coefficient according to a central wavelength of a light intensity peak or a light intensity valley and a pixel offset includes:

[0106] S710, taking the central wavelength of the light intensity peak or the light intensity valley as the second dependent variable, and taking the pixel offset as the second independent variable;

[0107] S720: Perform fitting according to the second dependent variable, the second independent variable and a preset second polynomial to determine a second calibration coefficient.

[0108] It should be noted that the second polynomial is determined according to actual application, and this embodiment does not impose any specific restrictions, such as multiple power polynomials or exponential polynomials, etc. During the fitting process, the stop condition is determined according to the objective function, such as fitting according to the least squares method.

[0109] In order to reduce the cost of calibration and avoid damaging the highly sensitive single-molecule fluorescence camera, fluorescent microspheres are used as the light source and calibration is performed through a four-channel filter block. In a specific embodiment, the specific steps are as follows:

[0110] Step S21: photographing the second image of the fluorescent microspheres in the imaging mode and the second spectral band in the spectral mode respectively.

[0111] Step S22: For the second image in the imaging mode, mark the position of the fluorescent microspheres.

[0112] Step S23: Perform two-dimensional Gaussian fitting on the data in the 20×20 pixel area around the marked position, so as to obtain a more accurate second spatial position coordinate (x im2 ,y im2 ),like Figure 7 As shown in (a).

[0113] Step S24: The result of the image distortion characterization and the second spatial position coordinate (x im2 ,y im2 ) into formula (1) to calculate the reference coordinates (x sp2 ,y sp2 ).

[0114] Step S25: For the second image in the spectral mode, take out the second reference coordinate (x sp2 ,y sp2 ) The data in the 20×110 pixel area around Figure 7 As shown in (b). Figure 7 As shown in (c), the light intensity profile along the Y direction is obtained by adding along the X direction. There are 4 peaks and 3 valleys in the light intensity profile, which correspond to the 4 passbands and 3 cutoff bands of the four-channel filter block. The two peaks on the far left and the far right are removed, and the data in the middle 2 peaks and 3 valleys are fitted with a Gaussian function to determine the center pixel (y' sp ). The center pixel (y' sp ) and the Y component of the second reference coordinate (y sp2 ) is subtracted to obtain the pixel offset (Δy sp Each peak and valley can be assigned a pixel offset (Δy sp ).

[0115] Step S26: Calculate the central wavelength λ. Each peak or valley corresponds to the central wavelength (λ) of the passband or cutoff band of the four-channel filter block, thereby obtaining 5 pairs of data points (λ, Δy sp ).

[0116] Step S27: Repeat steps S21 to S25 to calculate the pixel offsets (Δy sp ) and the corresponding wavelength (λ). For each passband or cutoff band, the average value of the pixel offset is calculated.

[0117] Step S28: Finally, use a third-order polynomial (Formula (2)) to fit the average value of the pixel offset The polynomial coefficients obtained from the curve of wavelength (λ) are the results of wavelength-pixel relationship calibration, such as Figure 7 As shown in (d).

[0118]

[0119] Among them, c3, c2, c1, and c0 are all coefficients to be determined.

[0120] In a specific embodiment, the calibration process of the single-molecule fluorescence spectrum imaging device is described. First, image distortion calibration is performed, and then wavelength-pixel relationship calibration is performed. The specific process is as follows.

[0121] In the imaging mode, a first image of the microsphere is collected, and the first spatial position coordinates are calculated according to the first image; in the spectral mode, a first spectral band of the microsphere is collected, and the first reference coordinates are calculated according to the first spectral band; the first spatial position coordinates and the first reference coordinates are fitted with a binary second-order polynomial to obtain a first calibration coefficient; in the imaging mode, a second image of the microsphere is collected, and the second spatial position coordinates are calculated according to the second image; the second reference coordinates are calculated according to the second spatial position coordinates, the binary second-order polynomial and the first calibration coefficient; in the spectral mode, a second spectral band of the microsphere is collected, and the center coordinates are calculated according to the second spectral band; a pixel offset is calculated according to the second reference coordinates and the center coordinates, and a third-order polynomial is fitted according to the pixel offset and the center wavelength to obtain a second calibration coefficient.

[0122] The implementation of the embodiment of the present invention includes the following beneficial effects: using the fluorescence emitted by the microsphere as the incident light, combined with a multi-channel filter block, to determine the pixel offset according to the center coordinates of the light intensity peak or the light intensity valley according to the second spectral band and the calibrated second reference coordinates, determining the second calibration coefficient according to the center wavelength of the light intensity peak or the light intensity valley in the second spectral band and the pixel offset, accurately mapping the pixel to the wavelength, and not needing to use multiple lasers as a reference for wavelength-pixel relationship calibration, lowering the cost and minimizing the risk of camera damage.

[0123] The following uses several specific embodiments to illustrate the application of the calibration device and method for a single-molecule fluorescence spectral imaging device.

[0124] Test Example 1: Measuring single-molecule fluorescence spectra

[0125] The following describes in detail the process of measuring the fluorescence spectrum of a single molecule of Cy5. Cy5 is a widely used fluorescent molecule with an emission peak wavelength of about 670 nanometers. First, a video is recorded in the imaging mode to locate the spatial position coordinates of a single molecule, such as Fig. 9 Then, switch to the spectral mode to collect spectral data, as shown in (a). Fig. 9 As shown in (b). Fig. 9 The single-step bleaching event observed in (c) can confirm that the measured fluorescence comes from a single molecule. In imaging mode, the position of the Cy5 molecule is first marked, and then the data of the surrounding 6×6 pixel area is taken out, and more accurate spatial position coordinates are obtained by fitting with a two-dimensional Gaussian function. Then the corresponding reference coordinates in the spectral mode are determined using the results of image distortion correction (Formula (1)). In order to obtain the spectral profile, the data of a 6×30 pixel area is intercepted with the reference coordinate as the center, and the pixel values ​​are summed along the X-axis to generate an intensity-pixel curve. The curve is converted into a spectrum using the third-order polynomial obtained in the wavelength-pixel relationship calibration (Formula (2)). The spectrum of a single Cy5 molecule is shown in Fig. 9 As shown in (d).

[0126] Test Example 2: Verifying the full-field spectral measurement capability

[0127] Existing similar devices lack the characterization of image distortion, resulting in the spectral measurement range being limited to a small range in the center of the field of view, and the measurement results outside this range are inaccurate. In order to prove that the device of the embodiment of the invention can accurately measure the single-molecule fluorescence spectrum within the entire field of view, the spectra before and after image distortion correction are compared, such as Fig.10 The entire field of view is divided into nine regions, and the spectral peak wavelength of the Cy5 molecule in each region is measured, as shown in Fig.10 The test results are shown in Fig.10In (b), without image distortion correction, the peak wavelength distributions in the nine regions vary greatly, and only the average value of the central region (i.e., region 5) is consistent with the expected peak wavelength of Cy5; wherein the hollow circles are the wavelength peak histogram counts without image distortion correction, and the blue line is the normal distribution fit of the data; the solid prisms are the wavelength peak histogram counts after image distortion correction, and the red line is the normal distribution fit of the data; the black vertical line is the theoretical value of the Cy5 peak wavelength (approximately 670 nanometers). In contrast, after correcting the image distortion, the peak wavelength distributions of all nine regions become highly consistent, and the average values ​​are close to the expected peak wavelength. Combining the data of all nine regions and statistically analyzing the peak wavelength distribution in the entire field of view, similar conclusions were obtained, such as Fig.10 As shown in (c). Without image distortion correction, the average peak wavelength of the distribution is 614.7 nanometers, which is significantly deviated from the expected emission peak of Cy5 (about 670 nanometers) and exhibits a large standard deviation of 40.4 nanometers. After image distortion correction, the peak wavelength distribution is concentrated around 668.7 nanometers, and the standard deviation is much smaller at 4.2 nm. These results collectively indicate that the calibration method of the single-molecule fluorescence spectroscopy imaging device in this embodiment is capable of achieving consistent and reliable single-molecule spectral measurements and peak wavelength determination across the entire field of view.

[0128] Test Example 3: Resolving power of different types of fluorescent molecules

[0129] One application of the single-molecule fluorescence spectral imaging device is to identify single molecules using their unique spectral "fingerprints". This requires the device to have a high enough spectral resolution to distinguish between two fluorescent molecules with closely adjacent spectral peak wavelengths. To test the spectral peak resolution of the device, a mixture of Cy3 and TAMRA, two fluorescent molecules with close emission peaks (570nm and 580nm, respectively), was analyzed. By measuring their spectral peak wavelengths, such as Fig.11 As shown in (b), the two fluorescent molecules were successfully distinguished. The time trajectory of the spectral peak wavelength shows that the standard deviation is less than 2nm ( Fig.11 Figure 2 (c) shows that the spectral peak wavelength resolution of the device is better than 2 nm, which is expected to meet the needs of most applications.

[0130] See also Fig.12 , an embodiment of the present invention provides a calibration device for a single-molecule fluorescence spectrum imaging device, comprising:

[0131] at least one processor;

[0132] at least one memory for storing at least one program;

[0133] When the at least one program is executed by the at least one processor, the at least one processor implements the above method.

[0134] Among them, the memory, as a non-transient computer-readable storage medium, can be used to store non-transient software programs and non-transient computer executable programs. The memory may include a high-speed random access memory, and may also include a non-transient memory, such as at least one disk storage device, a flash memory device, or other non-transient solid-state storage device. In some embodiments, the memory may optionally include a remote memory remotely arranged relative to the processor, and these remote memories may be connected to the processor via a network. Examples of the above-mentioned network include, but are not limited to, the Internet, an intranet, a local area network, a mobile communication network, and combinations thereof.

[0135] It can be seen that the contents of the above method embodiments are all applicable to the present device embodiments, the functions specifically implemented by the present device embodiments are the same as those of the above method embodiments, and the beneficial effects achieved are also the same as those achieved by the above method embodiments.

[0136] In addition, the embodiment of the present application also discloses a computer program product or a computer program, which is stored in a computer-readable storage medium. The processor of the computer device can read the computer program from the computer-readable storage medium, and the processor executes the computer program, so that the computer device performs the above method.

[0137] The embodiment of the present invention further provides a computer-readable storage medium, which stores a program executable by a processor, and the program executable by the processor is used to implement the above method when executed by the processor. Similarly, the contents of the above method embodiment are all applicable to the present storage medium embodiment, and the functions specifically implemented by the present storage medium embodiment are the same as those of the above method embodiment, and the beneficial effects achieved are also the same as those achieved by the above method embodiment.

[0138] It is understood that all or some steps and systems in the disclosed method above can be implemented as software, firmware, hardware and appropriate combinations thereof. Some physical components or all physical components can be implemented as software executed by a processor, such as a central processing unit, a digital signal processor or a microprocessor, or implemented as hardware, or implemented as an integrated circuit, such as an application-specific integrated circuit. Such software can be distributed on a computer-readable medium, and the computer-readable medium can include a computer storage medium (or a non-transitory medium) and a communication medium (or a temporary medium). As known to those of ordinary skill in the art, the term computer storage medium includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing information (such as computer-readable instructions, data structures, program modules or other data). Computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical disk storage, magnetic cassette, magnetic tape, disk storage or other magnetic storage device, or any other medium that can be used to store desired information and can be accessed by a computer. Furthermore, it is well known to those skilled in the art that communication media typically embodies computer readable instructions, data structures, program modules, or other data in a modulated data signal such as a carrier wave or other transport mechanism, and may include any information delivery media.

[0139] The above is a specific description of the preferred implementation of the present invention, but the invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A single-molecule fluorescence spectral imaging device, characterized in that: The invention comprises an aperture, a first lens, a first reflector module, a dispersion prism, a second reflector module, a second lens and a camera, wherein the first reflector module and the second reflector module can be moved into or out of the optical path at the same time; if the first reflector module and the second reflector module are moved into the optical path at the same time, an incident light passes through the aperture, the first lens, the first reflector module, the dispersion prism, the second reflector module and the second lens in sequence, and then forms an image on the camera; if the first reflector module and the second reflector module are moved out of the optical path at the same time, an incident light passes through the aperture, the first lens and the second lens in sequence, and then forms an image on the camera.

2. The single-molecule fluorescence spectral imaging device according to claim 1, characterized in that: The aperture comprises a two-dimensionally adjustable aperture.

3. The single-molecule fluorescence spectrum imaging device according to claim 1, characterized in that: The aperture and the camera satisfy a conjugate relationship.

4. The single-molecule fluorescence spectrum imaging device according to claim 1, characterized in that: The imaging device also includes a light source, a filter block and an objective lens. The light emitted by the light source passes through the filter block and the objective lens in sequence to reach the sample to be tested, so that the sample to be tested emits fluorescence, and the fluorescence passes through the filter block to form incident light.

5. A calibration method for a single-molecule fluorescence spectroscopy imaging device, characterized in that: The imaging device according to any one of claims 1 to 4, comprising: Simultaneously moving the first reflector module and the second reflector module out of the light path, acquiring a first image of the microsphere, and determining a first spatial position coordinate of the microsphere according to the first image; Simultaneously moving the first reflector module and the second reflector module into the optical path, acquiring a first spectral band of the microsphere, and determining a first reference coordinate according to the first spectral band; A first calibration coefficient is determined based on the first spatial position coordinates and the first reference coordinates.

6. The calibration method according to claim 5, characterized in that: The method further comprises: Simultaneously moving the first reflector module and the second reflector module out of the light path, acquiring a second image of the microsphere, and determining a second spatial position coordinate of the microsphere according to the second image; Simultaneously moving the first reflector module and the second reflector module into the optical path, acquiring a second spectral band of the microsphere, and determining the center coordinates of a light intensity peak or a light intensity valley according to the second spectral band; Determine a second reference coordinate according to the second spatial position coordinate and the first calibration coefficient, and determine a pixel offset according to the center coordinate and the second reference coordinate; The second calibration coefficient is determined according to the central wavelength of the light intensity peak or the light intensity valley and the pixel offset.

7. The calibration method according to claim 5, characterized in that: The determining a first calibration coefficient according to the first spatial position coordinate and the first reference coordinate comprises: Taking the first reference coordinate as a first dependent variable and the first spatial position coordinate as a first independent variable; A first calibration coefficient is determined by performing fitting according to the first dependent variable, the first independent variable and a preset first polynomial.

8. The calibration method according to claim 6, characterized in that: The determining of the second calibration coefficient according to the central wavelength of the light intensity peak or the light intensity valley and the pixel offset comprises: Taking the central wavelength of the light intensity peak or light intensity valley as a second dependent variable, and taking the pixel offset as a second independent variable; A second calibration coefficient is determined by performing fitting according to the second dependent variable, the second independent variable and a preset second polynomial.

9. The calibration method according to claim 6, characterized in that: The determining the pixel offset according to the center coordinate and the second reference coordinate comprises: respectively calculating a plurality of differences between the central coordinates of a plurality of light intensity peaks or light intensity valleys and the second reference coordinates; An average value of several of the difference values ​​is determined as the pixel shift.

10. A calibration device for a single-molecule fluorescence spectroscopy imaging device, characterized in that: include: at least one processor; at least one memory for storing at least one program; When the at least one program is executed by the at least one processor, the at least one processor implements the method according to any one of claims 5 to 9.

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