Rotating Interferometric Imaging Spectroscopic Microscopy System and Apparatus

Abstract

This invention provides a rotating interferometric imaging spectroscopic microscopy system and apparatus. Light emitted from a multi-band LED light source passes through a multi-band fluorescence filter to obtain narrowband light, which then enters an oil immersion lens to illuminate a fluorescently labeled sample. The excitation light from the fluorescently labeled sample passes through the multi-band fluorescence filter into a rotating Sagnac interferometer, forming two transversely sheared interference beams. These beams are then converged through a Fourier lens onto the image plane of a high-sensitivity camera, generating an interference image. The high-sensitivity camera continuously acquires multiple frames of interference images. A computer processing module performs multi-frame interference image registration and Fourier transform to achieve spectral reconstruction, obtaining a unified spectral data cube that includes both two-dimensional image information and one-dimensional spectral information of the fluorescently labeled sample. This invention utilizes interferometry to obtain the spectral information of all pixels in the sample, offering high light throughput and high reliability, thus improving the imaging system's resolution of fluorescently labeled samples.

Description

Technical Field

[0001] This invention relates to the field of interferometric imaging spectral microscopy, and more specifically, to a rotating interferometric imaging spectral microscopy system and apparatus. Background Technology

[0002] In existing biomedical imaging analysis techniques, fluorescent labeling is the mainstream technology. Fluorescently labeled probes specifically bind to target component molecules, allowing researchers to observe the fluorescence under a fluorescence microscope and thus detect the presence of the target component. Samples hybridized with fluorescently labeled probes become fluorescently labeled samples. With the increasing demand for precise localization, qualitative, and quantitative analysis of samples, the currently widely used method of visually observing fluorescent samples through a microscope is insufficient. Firstly, the excitation fluorescence of fluorescently labeled samples is relatively weak, making it difficult to detect and susceptible to interference from illumination. Secondly, the human eye can only observe the color and texture information of a two-dimensional image of a sample; confusion can easily arise with fluorescent labels of similar colors, and misjudgment can easily occur with overlapping fluorescent labels.

[0003] Interferometric spectroscopy imaging combines imaging and interferometric spectroscopy techniques. It generates two coherent beams as a reference and a measurement beam, which then interfere with each other. The resulting interference fringes reflect the optical path difference between the reference and measurement beams. The transformation of these interference fringes allows for the derivation of the spectral information of the fluorescently labeled sample. Spectral information is a powerful complement to color and texture information, and spectral characteristics can effectively reflect changes in the physicochemical properties of the sample.

[0004] Fourier transform interferometric imaging spectrometers are machines based on interferometric spectral imaging technology. Compared with traditional dispersive imaging spectrometers, interferometric imaging spectrometers have advantages such as high spectral resolution, high light flux, and high signal-to-noise ratio. Fourier transform interferometric imaging spectrometers have developed rapidly in the last fifty years and have been widely used in the biomedical field.

[0005] Of the three types of Fourier transform interferometric imaging spectrometers, the time-modulated type has the highest resolution and sensitivity. Time-modulated Fourier transform spectrometers require a large optical path difference to be generated through a push-broom mechanism, necessitating a precise moving mirror push-broom system with high requirements for motion control and vibration damping performance. Introducing the optical path difference using a rotating structure, instead of the linear or oscillating moving mirror push-broom structure, can improve system stability, detection sensitivity, and detection speed.

[0006] The rotating mirror high-sensitivity interferometric spectral imager (ROSI) proposed by Yuan Yan et al. obtains the spatial and spectral information of the target by rotating one of the reflective surfaces of the Sagnac interferometer (Design method of rotating mirror high-sensitivity interferometric spectral imager ROSI, Acta Photonica Sinica, 2007, 36(2):279-281). This method has strict limitations on the rotation angle, is not easy to assemble and adjust, and has poor overall stability.

[0007] The rotating Fourier transform interferometric imaging spectrometer proposed in Chinese Patent 102759402B obtains the optical path difference by rotating a cubic angle or a mirror. However, it only rotates some components, has a small adjustable angle, and has poor overall stability.

[0008] In summary, time-modulated Fourier transform spectrometers to date have suffered from problems such as poor stability, low efficiency in obtaining unified spectral images, and low light throughput. In the biomedical field, imaging of fluorescently labeled samples presents challenges such as susceptibility to interference from light sources and stray light, and confusion arising from excitation of similar colors. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the purpose of this invention is to provide a rotating interferometric imaging spectral microscopy system and device.

[0010] The rotating interferometric imaging spectral microscopy system provided by the present invention includes: a multi-band LED light source, a multi-band fluorescence filter box, an oil immersion lens, a stage scanning motion module, a rotating Sagnac interferometer, a Fourier lens, a high-sensitivity camera, and a computer processing module.

[0011] The light emitted by the multi-band LED light source passes through the multi-band fluorescent filter box to obtain narrowband light, which enters the oil immersion lens to illuminate the fluorescent label sample to be tested. The fluorescent label sample to be tested is placed on the stage scanning motion module.

[0012] The excitation light of the fluorescently labeled sample passes through a multi-band fluorescence filter box and enters a rotating Sagnac interferometer, forming two transversely sheared interference beams. These beams are then converged onto the image plane of a high-sensitivity camera through a Fourier lens to produce an interference image.

[0013] By rotating the Sagnac interferometer, the sample is continuously scanned, and multiple frames of interference images are continuously acquired by a high-sensitivity camera to form an interference data cube.

[0014] The high-sensitivity camera is connected to a computer processing module, which performs multi-frame interferometric image registration and Fourier transform to achieve spectral reconstruction, resulting in a spectral data cube that combines two-dimensional image information and one-dimensional spectral information of fluorescently labeled samples.

[0015] Preferably, the multi-band LED light source is adapted to the excitation band required by the fluorescent labeling probe used in the fluorescently labeled sample to be tested, and is composed of multiple monochrome LED chips. The center wavelength of its emission does not overlap with the center wavelength of the fluorescence generated by the probe when excited, and is shorter than the center wavelength of the fluorescence generated by the probe when excited.

[0016] Preferably, the multi-band fluorescence filter box is used to filter the multi-band combined light emitted by the multi-band LED light source, narrow the wavelength of the light reflected from the light source entering the oil immersion lens, and transmit light to the stimulated fluorescence generated by the fluorescently labeled sample.

[0017] The oil mirror faces the side of the multi-band fluorescence filter box. The light reflected from the multi-band fluorescence filter box enters the oil mirror and illuminates the fluorescently labeled sample located on the scanning motion module of the stage. The fluorescently labeled sample is excited by the multi-band reflected light transmitted through the oil mirror. The generated fluorescence passes through the oil mirror, through the multi-band fluorescence filter box, and enters the beam splitter prism in the rotating Sagnac interferometer.

[0018] Preferably, the rotating Sagnac interferometer includes a circular turntable, a beam-splitting prism, a first reflecting mirror, and a second reflecting mirror; the beam-splitting prism is fixed at the center of the circular turntable and is located in the direction of the transmitted light from the multi-band fluorescence filter box; the image light from the transmitted portion of the beam-splitting prism forms one path of transmitted light, and the image light from the reflected portion of the beam-splitting prism forms one path of reflected light.

[0019] The first reflector is located in one transmission direction of the beam splitter prism, and the second reflector is located in one reflection direction of the beam splitter prism.

[0020] Preferably, the first reflector and the beam splitter are located on a quasi-straight line in the Y direction, forming an angle of 67.5 degrees with the incident light direction.

[0021] Preferably, the second reflector and the beam splitter are located on a quasi-straight line in the X direction, forming an angle of 67.5 degrees with the direction of reflected light and an angle of 45 degrees with the first reflector.

[0022] Preferably, the Fourier lens is located in the direction of the beam-splitting prism's output light, parallel to the beam-splitting prism's output surface, with the center of the Fourier lens coinciding with the center of the principal ray, and the image plane of the Fourier lens coinciding with the sensor array of the high-sensitivity camera.

[0023] Preferably, the high-sensitivity camera is located on the principal optical axis of the Fourier lens.

[0024] Preferably, the computer processing module receives the signal from the high-sensitivity camera, processes the acquired interferometric data cube, and finally obtains a spectral data cube with integrated image and spectrum, including the following steps:

[0025] Step S1: Obtain the grayscale value sequence of the interference data from multiple frames of images;

[0026] Step S2: Fit the trend term using a quadratic polynomial, in the form: f(x) = a²x 2 +a1x+a0, where a0, a1, and a2 are coefficients to be determined. The least squares method is used to determine these coefficients so that the sum of squared errors between all data points and the polynomial function f(x) is minimized. The polynomial part is subtracted from the extracted interferometric data to remove linear or nonlinear trends in the data.

[0027] Step S3: Add a triangular window to the interferometric data. The mathematical definition of the triangular window is as follows: Let the length of the window function be N, then the definition of the triangular window ω(n) on n = 0, 1, ..., N-1 is: n is the index of the window function;

[0028] Step S4: Perform Fourier transform on the interference data to obtain the pixel spectrum. Repeat the above operation on a single pixel on the entire image to obtain the spectral data cube of the fluorescence sample.

[0029] The rotating interferometric imaging spectral microscope device provided by the present invention employs the aforementioned rotating interferometric imaging spectral microscope system.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention combines high numerical aperture high-magnification oil immersion microscopy and high-resolution interferometric spectral imaging techniques. The designed multi-band LED reduces interference from broadband light sources on fluorescence detection, and the designed multi-band fluorescence filter box efficiently filters light to further reduce interference. The system exhibits good overall integrity and high light throughput, directly obtaining a unified image and spectrum. It automatically acquires a user-defined number of unified image and spectrum datasets, obtaining interference images of samples under different optical path differences, and then retrieving the spectral distribution of each pixel in the sample. Attached Figure Description

[0032] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0033] Figure 1 This is a schematic diagram of the rotating interferometric imaging spectral microscopy system of the present invention;

[0034] Figure 2 This is a flowchart illustrating the processing of the acquired interferometric data cube. Detailed Implementation

[0035] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0036] Example

[0037] like Figure 1 The present invention provides a rotating interferometric imaging spectral microscopy system, comprising: a multi-band LED light source 1, a multi-band fluorescence filter box 2, an oil immersion lens 3, a stage scanning motion module 4, a rotating Sagnac interferometer 5, a Fourier lens 6, a high-sensitivity camera 7, and a computer processing module 8.

[0038] The light emitted by the multi-band LED light source 1 passes through the multi-band fluorescent filter box 2 to obtain narrowband light, and the narrowband light enters the oil immersion lens 3 to illuminate the fluorescent label sample to be detected. The fluorescent label sample to be detected is placed on the stage scanning motion module 4.

[0039] The excitation light of the fluorescently labeled sample passes through the multi-band fluorescence filter box 2 and enters the rotating Sagnac interferometer 5, forming two transversely sheared interference beams. These beams are then converged onto the image plane of the high-sensitivity camera 7 through the Fourier lens 6 to produce an interference image.

[0040] By rotating the Sagnac interferometer 5, the sample is continuously scanned, and multiple frames of interference images are continuously acquired by the high-sensitivity camera 7, forming an interference data cube.

[0041] The high-sensitivity camera 7 is connected to the computer processing module 8. The computer processing module 8 performs multi-frame interferometric image registration and Fourier transform to achieve spectral reconstruction, resulting in a spectral data cube that combines two-dimensional image information and one-dimensional spectral information of fluorescently labeled samples.

[0042] The multi-band LED light source 1 is adapted to the excitation band required by the fluorescent labeling probe used in the fluorescently labeled sample to be tested. It consists of multiple monochrome LED chips, and the center wavelength of its emission does not overlap with the center wavelength of the fluorescence generated by the probe when excited, and is shorter than the center wavelength of the fluorescence generated by the probe when excited, so as to excite the probe fluorescence.

[0043] The multi-band fluorescence filter box 2 is specially designed to finely filter the multi-band combined light emitted by the multi-band LED light source 1, narrow the wavelength of the light reflected from the light source entering the oil immersion 3, and has extremely high transmittance for the stimulated fluorescence generated by the fluorescent sample.

[0044] The oil mirror 3 faces the side of the multi-band fluorescence filter box 2. The light reflected by the multi-band fluorescence filter box 2 enters the oil mirror 3 and illuminates the fluorescently labeled sample located on the scanning motion module 4 of the stage. The fluorescently labeled sample is excited by the multi-band reflected light transmitted through the oil mirror 3. The fluorescence generated by the excitation passes through the oil mirror 3, through the multi-band fluorescence filter box 2, and enters the beam splitter prism in the rotating Sagnac interferometer 5.

[0045] The rotating Sagnac interferometer 5 includes a circular turntable, a beam splitter prism, a first reflecting mirror, and a second reflecting mirror. The beam splitter prism is fixed at the center of the circular turntable and is located in the direction of the transmitted light from the multi-band fluorescence filter box 2. The transmitted light from the beam splitter prism forms a transmitted light path, and the reflected light from the beam splitter prism forms a reflected light path.

[0046] The first reflector is located in one transmission direction of the beam splitter prism, and the second reflector is located in one reflection direction of the beam splitter prism.

[0047] The first reflecting mirror and the beam splitter are located on a quasi-straight line in the Y direction, forming an angle of 67.5 degrees with the incident light direction.

[0048] The second reflector and the beam splitter are located on a quasi-straight line in the X direction, forming an angle of 67.5 degrees with the direction of reflected light and an angle of 45 degrees with the first reflector.

[0049] The Fourier lens 6 is located in the direction of the beam-splitting prism's output light and is parallel to the beam-splitting prism's output surface. The center of the Fourier lens 6 coincides with the center of the principal ray, and the image plane of the Fourier lens 6 coincides with the photosensitive element array of the high-sensitivity camera 7.

[0050] The high-sensitivity camera 7 is located on the principal optical axis of the Fourier lens 6.

[0051] The computer processing module 8 receives signals from the high-sensitivity camera 7, processes the acquired interferometric data cube, and finally obtains a spectral data cube with integrated image and spectrum, such as... Figure 2 This includes the following steps:

[0052] Step S1: Obtain the grayscale value sequence of the interference data from multiple frames of images;

[0053] Step S2: Fit the trend term using a quadratic polynomial, in the form: f(x) = a²x 2 +a1x+a0, where a0, a1, and a2 are coefficients to be determined. The least squares method is used to determine these coefficients, minimizing the sum of squared errors between all data points and the polynomial function f(x), i.e., minimizing: The polynomial part is subtracted from the extracted interferometric data to remove linear or nonlinear trends from the data.

[0054] Step S3: Apodization. To mitigate the Gibbs phenomenon, a triangular window is added to the interferometric data. The mathematical definition of the triangular window is as follows: Let the length of the window function be N, then the definition of the triangular window ω(n) on n = 0, 1, ..., N-1 is: n is the index of the window function;

[0055] Step S4: Perform Fourier transform on the interference data to obtain the pixel spectrum. Repeat the above operation on a single pixel on the entire image to obtain the spectral data cube of the fluorescence sample.

[0056] The present invention also provides a rotating interferometric imaging spectral microscopy device, which employs the aforementioned rotating interferometric imaging spectral microscopy system.

[0057] In the description of this application, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0058] Those skilled in the art will understand that, in addition to implementing the system, apparatus, and their modules provided by this invention in purely computer-readable program code, the same program can be implemented in the form of logic gates, switches, application-specific integrated circuits, programmable logic controllers, and embedded microcontrollers by logically programming the method steps. Therefore, the system, apparatus, and their modules provided by this invention can be considered a hardware component, and the modules included therein for implementing various programs can also be considered structures within the hardware component; alternatively, modules for implementing various functions can be considered both software programs implementing the method and structures within the hardware component.

[0059] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A rotating interferometric imaging spectroscopic microscopy system, characterized in that, include: Multi-band LED light source (1), multi-band fluorescent filter box (2), oil immersion lens (3), stage scanning motion module (4), rotating Sagnac interferometer (5), Fourier lens (6), high-sensitivity camera (7) and computer processing module (8); The light emitted by the multi-band LED light source (1) passes through the multi-band fluorescent filter box (2) to obtain narrowband light, and the narrowband light enters the oil immersion lens (3) to irradiate the fluorescent label sample to be tested. The fluorescent label sample to be tested is placed on the stage scanning motion module (4). The excitation light of the fluorescently labeled sample passes through the multi-band fluorescence filter box (2) and enters the rotating Sagnac interferometer (5), forming two beams of transverse shear interference light, which are then converged onto the image plane of the high-sensitivity camera (7) through the Fourier lens (6) to produce an interference image; By rotating the Sagnac interferometer (5), the sample is continuously scanned, and multiple frames of interference images are continuously acquired by the high-sensitivity camera (7) to form an interference data cube; The high-sensitivity camera (7) is connected to the computer processing module (8). The computer processing module (8) performs multi-frame interference image registration and Fourier transform to achieve spectral reconstruction, resulting in a spectral data cube that combines two-dimensional image information and one-dimensional spectral information of fluorescently labeled samples.

2. The rotating interferometric imaging spectroscopic microscopy system according to claim 1, characterized in that, The multi-band LED light source (1) is adapted to the excitation band required by the fluorescent labeling probe used in the fluorescent labeling sample to be tested. It consists of multiple monochrome LED chips, and its center wavelength of emission does not overlap with the center wavelength of the fluorescence generated by the probe when excited, and is shorter than the center wavelength of the fluorescence generated by the probe when excited.

3. The rotating interferometric imaging spectroscopic microscopy system according to claim 1, characterized in that, The multi-band fluorescence filter box (2) is used to filter the multi-band combined light emitted by the multi-band LED light source (1), narrow the light band reflected from the light source entering the oil immersion (3), and transmit the stimulated fluorescence generated by the fluorescently labeled sample. The oil mirror (3) faces the multi-band fluorescence filter box (2). The light reflected by the multi-band fluorescence filter box (2) enters the oil mirror (3) and illuminates the fluorescently labeled sample located on the stage scanning motion module (4). The fluorescently labeled sample is excited by the multi-band reflected light transmitted through the oil mirror (3). The generated fluorescence passes through the oil mirror (3), through the multi-band fluorescence filter box (2), and enters the beam splitter in the rotating Sagnac interferometer (5).

4. The rotating interferometric imaging spectroscopic microscopy system according to claim 1, characterized in that, The rotating Sagnac interferometer (5) includes a circular turntable, a beam splitter prism, a first reflecting mirror, and a second reflecting mirror; the beam splitter prism is fixed at the center of the circular turntable and is located in the direction of the transmitted light of the multi-band fluorescence filter box (2); the image light of the transmitted part of the beam splitter prism forms a transmitted light path, and the image light of the reflected part of the beam splitter prism forms a reflected light path. The first reflector is located in one transmission direction of the beam splitter prism, and the second reflector is located in one reflection direction of the beam splitter prism.

5. The rotating interferometric imaging spectroscopic microscopy system according to claim 4, characterized in that, The first reflecting mirror and the beam splitter are located on a quasi-straight line in the Y direction, forming an angle of 67.5 degrees with the direction of incident light.

6. The rotating interferometric imaging spectroscopic microscopy system according to claim 5, characterized in that, The second reflector and the beam splitter are located on a quasi-straight line in the X direction, forming an angle of 67.5 degrees with the direction of reflected light and an angle of 45 degrees with the first reflector.

7. The rotating interferometric imaging spectroscopic microscopy system according to claim 4, characterized in that, The Fourier lens (6) is located in the direction of the beam-splitting prism's output light and is parallel to the beam-splitting prism's output surface. The center of the Fourier lens (6) coincides with the center of the principal ray, and the image plane of the Fourier lens (6) coincides with the photosensitive element array of the high-sensitivity camera (7).

8. The rotating interferometric imaging spectroscopic microscopy system according to claim 1, characterized in that, The high-sensitivity camera (7) is located on the principal optical axis of the Fourier lens (6).

9. The rotating interferometric imaging spectroscopic microscopy system according to claim 1, characterized in that, The computer processing module (8) receives the signal from the high-sensitivity camera (7), processes the acquired interferometric data cube, and finally obtains a spectral data cube with integrated image and spectrum, including the following steps: Step S1: Obtain the grayscale value sequence of the interference data from multiple frames of images; Step S2: Fit the trend term using a quadratic polynomial, in the form: f(x) = a²x 2 +a1x+a0, where a0, a1, and a2 are coefficients to be determined. The least squares method is used to determine these coefficients so that the sum of squared errors between all data points and the polynomial function f(x) is minimized. The polynomial part is subtracted from the extracted interferometric data to remove linear or nonlinear trends in the data. Step S3: Add a triangular window to the interferometric data. The mathematical definition of the triangular window is as follows: Let the length of the window function be N, then the definition of the triangular window ω(n) on n = 0, 1, ..., N-1 is: n is the index of the window function; Step S4: Perform Fourier transform on the interference data to obtain the pixel spectrum. Repeat the above operation on a single pixel on the entire image to obtain the spectral data cube of the fluorescence sample.

10. A rotating interferometric imaging spectroscopic microscope, characterized in that, The rotating interferometric imaging spectroscopic microscopy system according to any one of claims 1 to 9 is used.

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