Hyperspectral microscope

Abstract

The invention relates to the technical field of optical microscopic imaging and spectrum detection, and discloses a hyperspectral microscope. The microscope comprises a microscope objective, an optical splitter, a tube lens, an image plane and an incident spectrum light source, the microscope objective adopts an apochromatic objective, the optical splitter is of a semi-transparent and semi-reflective structure, the tube lens is of an achromatic type, and the image plane integrates a signal processing circuit and a high-speed data interface. The incident spectrum light source has the capability of outputting monochromatic light beams with high spectral resolution; the equipment is equipped with an automatic control module and a calibration module, and core components are fixed on the same optical platform. The device overcomes the defects of chromatic aberration, light loss, poor stability and the like of existing equipment, realizes high-precision and high-efficiency acquisition of microscopic morphology and hyperspectral data of an observed object, and is suitable for microscopic spectral analysis in the fields of biomedicine, material science and the like.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy imaging and spectral detection technology, and in particular to a hyperspectral microscope for microscopic observation of objects and high-precision acquisition of their hyperspectral image data. Background Technology

[0002] As a core device integrating microscopic imaging technology and spectral detection technology, the hyperspectral microscope can simultaneously acquire the microscopic morphology information and spectral characteristic data of the observed object, realizing the simultaneous analysis of morphological visualization and composition quantification. It has irreplaceable application value in fields such as biomedicine, materials science, and microelectronics detection.

[0003] Existing hyperspectral microscopes typically consist of a microscope objective, an illumination system, a spectrometer, an imaging unit, and a data processing unit. Their basic working principle is to provide a broadband or monochromatic light beam through the illumination system to illuminate the object being observed. After being magnified by the microscope objective, the light signals of different wavelengths are separated by the spectrometer. The image data is then acquired by the imaging unit, and finally, the data processing unit generates a hyperspectral data cube.

[0004] However, existing hyperspectral microscopes have many technical shortcomings in practical applications, which severely limit their detection accuracy and scope of application: Insufficient chromatic aberration correction in objectives: Existing technologies mostly use ordinary achromatic objectives or semi-apochromatic objectives, which can only correct chromatic aberration and spherical aberration for some wavelengths. However, hyperspectral imaging requires imaging at multiple consecutive wavelengths. Uncorrected chromatic aberration will cause the image plane position of light signals of different wavelengths to shift, making it impossible to accurately align the images corresponding to each wavelength. This directly causes matching errors between spectral data and spatial position, affecting the accuracy of component analysis.

[0005] Performance defects of beam splitting components: Most existing beam splitters adopt thin-film structures, which are prone to angular deviations during assembly and adjustment, resulting in uneven beam splitting ratios. At the same time, the transmittance and reflectance of thin-film beam splitters are greatly affected by wavelength, and there is significant optical signal attenuation in some bands. The optical coupling efficiency is less than 50%, resulting in weak imaging light intensity and low signal-to-noise ratio.

[0006] Unreasonable optical path stability and layout: The core components of existing equipment, such as microscope objectives, beam splitters, and tube lenses, are mostly installed in a decentralized manner, lacking a unified fixed reference. They are easily affected by environmental vibrations or temperature changes, resulting in positional shifts. At the same time, the optical path design does not fully consider the parameter matching between components. For example, the focal length of the tube lens does not match the magnification of the objective lens, and the numerical aperture of the condenser lens does not match the objective lens. This leads to a limited imaging field of view and a decrease in resolution.

[0007] Limited illumination methods: Existing equipment mostly uses single reflective or transmissive illumination, which cannot be flexibly switched according to the characteristics of the observed object (such as transparent / opaque, biological slices / thin film materials). This results in poor illumination uniformity for some types of observed objects, making it impossible to effectively measure the observed target.

[0008] Lack of automation and calibration mechanisms: Existing equipment relies heavily on manual operation for focusing, wavelength switching, and image acquisition, resulting in low acquisition efficiency and the potential for errors due to manual operation. In addition, the lack of a regular calibration module means that as the equipment is used for a long time, issues such as optical path offset and light source wavelength drift will lead to a continuous decline in data accuracy, failing to meet the long-term high-precision detection requirements.

[0009] These shortcomings make it difficult for existing hyperspectral microscopes to meet practical application needs in terms of high-precision spectral imaging, multi-scene adaptation, and efficient data acquisition, thus limiting their promotion in high-end scientific research and industrial testing fields. Summary of the Invention

[0010] The present invention addresses at least one of the following technical problems: the chromatic aberration of the objective lens in existing hyperspectral microscopes leads to low spectral imaging accuracy; the optical coupling efficiency of the beam splitter is poor and the stability is insufficient; the unreasonable optical path layout leads to poor stability; the single illumination method cannot be adapted to different observation objects; and the lack of automated control and calibration mechanisms leads to low acquisition efficiency and insufficient data accuracy.

[0011] To address the aforementioned technical problems, this invention provides a hyperspectral microscope to achieve high-precision, high-stability, and multi-scene adaptability hyperspectral image data acquisition. Specifically: A hyperspectral microscope is characterized by comprising a microscope objective, a beam splitter, a tube mirror, and an image plane. The microscope objective is used to magnify and image the observed object and collect light signals. The beam splitter is disposed in the image-side optical path of the microscope objective. The tube mirror is disposed in the optical path of the beam splitter on the side away from the microscope objective. The image plane is disposed at the image-side focal point of the tube mirror and is used to receive light signals and convert them into digital image signals.

[0012] In a preferred embodiment of the present invention, the microscope objective is an infinity-corrected apochromatic objective whose spectral response range covers the operating wavelength range of the incident spectral light source.

[0013] In a preferred embodiment of the present invention, the beam splitter is a semi-transparent and semi-reflective beam splitter prism or a transparent thin sheet coated with a beam splitter film. Its light-transmitting surface is set at a preset angle with the outgoing light path of the microscope objective, which enables the outgoing light from the microscope objective to be incident perpendicularly and transmitted along the original light path extension direction, while allowing the outgoing light from the incident spectral light source to be reflected into the light path of the microscope objective.

[0014] In a preferred embodiment of the present invention, the size and aperture of the beam splitter are adapted to the output spot size of the microscope objective; if it is a cubic beam splitter prism, its bonding surface is bonded with optical adhesive, which has good flatness; if it is a beam splitter diaphragm, it is fixed in a metal frame with an adjustment mechanism, and the size and aperture of the metal frame are adapted to the output spot size of the microscope objective.

[0015] In a preferred embodiment of the present invention, the light source further includes an incident spectral light source, which is a monochromatic beam with high spectral resolution. Its outgoing light path is directed toward the reflective surface of the beam splitter, or it directly illuminates the object of observation through an external optical system, or it illuminates the object of observation invertedly through an external optical system.

[0016] In a preferred embodiment of the present invention, the incident spectral light source is a monochromatic light source formed by a tunable laser, a monochromator, and a broadband light source, or an array of monochromatic LED light sources; the slit width of the monochromator is adjustable to balance spectral resolution and light intensity; the array of monochromatic LED light sources consists of multiple LEDs emitting different wavelengths, and different wavelengths of illumination are achieved by sequentially lighting different LEDs.

[0017] In a preferred embodiment of the present invention, the tube is an infinity-corrected achromatic tube, whose object-side focal point coincides with the image-side focal point of the microscope objective, and whose focal length matches the magnification of the microscope objective. It can correct chromatic aberration during the transmission of light signals, and its aperture is adapted to the size of the exit spot of the microscope objective.

[0018] In a preferred embodiment of the present invention, the image plane is a CCD or CMOS image sensor, which integrates a signal processing circuit for noise reduction and amplification of electrical signals, and is connected to the image center processing system through a data transmission interface.

[0019] In a preferred embodiment of the present invention, an automatic control module and a calibration module are also included. The automatic control module is electrically connected to the incident spectral light source, the image plane, the stage, and the focusing mechanism to realize automated hyperspectral image acquisition. The calibration module includes a standard reference sample and a calibration control unit to calibrate the optical path and spectral accuracy of the device.

[0020] In a preferred embodiment of the present invention, a light shield and a focusing mechanism are also included. The light shield is disposed on the outer side of the object side of the microscope objective and around the stage to block interference from ambient light. The focusing mechanism is used to adjust the distance between the microscope objective and the object being observed in order to achieve clear imaging.

[0021] Compared with the prior art, the present invention has the following significant advantages: This invention employs an apochromatic objective lens, which uses a lens group composed of multiple low-dispersion optical glasses to effectively correct chromatic aberration and spherical aberration at multiple wavelengths, thus solving the problem of image plane offset in existing objectives; in conjunction with an apochromatic tube lens, it ensures accurate imaging of light signals of different wavelengths on the image plane.

[0022] The beam splitter can be a cubic beam splitter prism or an adjustable angle beam splitter film. The flatness error of the cemented surface of the cubic beam splitter prism is less than one-quarter wavelength, avoiding assembly and adjustment errors and ensuring a uniform beam splitting ratio. The anti-reflection / anti-transmittance film has high transmittance and reflectance in the target band, low optical loss, and significantly improved coupling efficiency. The core components are integrated, resulting in strong optical path stability.

[0023] The light source is compatible with various types, has high spectral resolution, and can capture subtle spectral characteristics; it covers visible light and a wider band, and the slit / LED adjustment can balance resolution and light intensity to meet diverse needs.

[0024] Three lighting modes can be flexibly selected according to sample type, with precise field of view matching, high utilization rate, and solving the problem of single lighting mode.

[0025] FPGA automatic control supports wavelength setting, high-precision focusing, and continuous acquisition, with high efficiency and no need for manual intervention; standard sample calibration is highly accurate, can correct offset drift, ensure long-term data accuracy, and has extremely low error.

[0026] Scientific-grade CCD / CMOS sensors with high pixel count and high quantum efficiency, combined with signal processing circuitry to improve the signal-to-noise ratio; support high-speed transmission, and combined with processing algorithms to quickly generate analysis images with high analysis efficiency. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the optical path structure of a hyperspectral microscope provided in the first embodiment of the present invention.

[0028] The labels for each figure are as follows: 1: microscope objective, 2: beam splitter, 3: tube lens, 4: image plane. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0031] like Figure 1As shown, the first embodiment of the present invention provides a hyperspectral microscope suitable for microscopic observation of objects and acquisition of their hyperspectral image data. Figure 1 As shown, the hyperspectral microscope includes a microscope objective 1, a beam splitter 2, a tube mirror 3, and an image plane 4.

[0032] The microscope objective 1 is the imaging front-end component of the hyperspectral microscope. Its function is to magnify and image the observed object to obtain its microscopic morphological information. Preferably, the microscope objective 1 is an infinity-corrected objective lens, which has a high numerical aperture and magnification. It can collect the light signals reflected or transmitted from the surface of the observed object while ensuring imaging resolution, providing a clear initial image for subsequent spectral imaging. Furthermore, the object-side focal point of the microscope objective 1 corresponds to the placement position of the observed object; that is, the observed object is positioned within the object-side working distance range of the microscope objective 1 to ensure that the observed object can be clearly imaged by the microscope objective 1.

[0033] Furthermore, the magnification of the microscope objective 1 can be selected according to the size of the object being observed and the observation requirements. Non-limitingly, the magnification can be 10x, 20x, 40x or 100x, etc.

[0034] Preferably, the spectral response range of the microscope objective 1 can cover the working wavelength range of the incident spectral light source. For example, if the working wavelength range of the incident spectral light source is the visible light region from 400 nm to 760 nm, then the microscope objective 1 can have stable imaging quality and transmittance within this wavelength range to avoid imaging distortion or light intensity loss at different wavelengths.

[0035] In a preferred embodiment of the present invention, the microscope objective 1 is an apochromatic objective. This type of objective can correct chromatic aberration and spherical aberration at multiple wavelengths, ensuring that light signals of different wavelengths can form clear images after passing through the microscope objective 1. This is particularly important for hyperspectral imaging, because hyperspectral imaging requires imaging at multiple wavelengths. If the objective has chromatic aberration, it will cause the image plane 4 at different wavelengths to shift, thereby affecting the accuracy of hyperspectral image data. Furthermore, the lens group of the apochromatic objective is made of a variety of low-dispersion optical glasses. By complementing the dispersion characteristics of different glass materials, chromatic aberration correction is achieved. Its imaging distortion is less than 0.1% across the entire spectrum, which can meet the high-precision requirements of hyperspectral imaging.

[0036] The beam splitter 2 is a semi-transparent, semi-reflective beam splitting prism or beam splitting film, which is disposed in the image-side optical path of the microscope objective 1. Specifically, one of the light-transmitting surfaces of the beam splitter 2 is set at a preset angle to the exit optical path of the microscope objective 1, preferably 45 degrees, so that the exit light from the microscope objective 1 can be perpendicularly incident on the light-transmitting surface of the beam splitter 2 and continue to propagate along the extension direction of the original optical path, while allowing the exit light from the incident spectral source to be reflected by the beam splitter 2 into the optical path of the microscope objective 1. Furthermore, the size and aperture of the beam splitter 2 are adapted to the exit spot size of the microscope objective 1 to avoid optical signal loss and ensure effective coupling of illumination light and imaging light.

[0037] Furthermore, the splitting ratio of the beam splitter 2 is selected based on the light intensity of the incident spectral light source and the light response characteristics of the image plane 4. Preferably, the transmittance and reflectance of the beam splitter 2 are both not less than 50% to ensure that the imaging optical path can obtain sufficient light signal, while the illumination optical path can also provide sufficient illumination intensity. The surface of the beam splitter 2 is usually coated with an antireflection film or an antireflection film to improve the transmittance or reflectance within a specific wavelength range. For example, in a hyperspectral microscope operating in the visible light region, the antireflection film and antireflection film of the beam splitter 2 need to have high optical performance in the range of 400nm to 760nm, with a transmittance and reflectance of not less than 90%, in order to reduce the loss of light signal.

[0038] In one illustrative embodiment of the present invention, the beam splitter 2 is a cubic beam splitter prism. This type of beam splitter prism has a flat light-transmitting surface, a uniform beam splitting ratio, and can avoid the assembly and adjustment errors of the diaphragm-type beam splitter 2. Accordingly, the size of the cubic beam splitter prism must be adapted to the size of the exit spot of the microscope objective 1. For example, if the diameter of the exit spot of the microscope objective 1 is 10 mm, then the aperture of the cubic beam splitter prism must be no less than 10 mm to ensure that the light signal can pass through completely. Furthermore, the bonding surface of the cubic beam splitter prism is bonded with optical adhesive to avoid light signal distortion caused by uneven bonding surfaces and to ensure the stability of the optical path.

[0039] In another embodiment of the present invention, the beam splitter 2 employs a beam-splitting diaphragm. This type of beam-splitting diaphragm is relatively thin, which saves optical path space and is suitable for scenarios with limited optical path space. The beam-splitting diaphragm is fixed within a metal frame. The size of the metal frame and its aperture are adapted to the size of the emitted light spot of the microscope objective 1. Simultaneously, the metal frame has an adjustment mechanism that can adjust the angle of the beam-splitting diaphragm to ensure that it forms a 45-degree angle with the emitted light path of the microscope objective 1.

[0040] Optionally, the hyperspectral microscope further includes an incident spectral light source. Preferably, the incident spectral light source is a monochromatic light source with high spectral resolution. Non-limitingly, the incident spectral light source can be a monochromatic light source formed by a tunable laser, a monochromator combined with a broadband light source, or an array of monochromatic LED light sources, etc. Further, the outgoing light path of the incident spectral light source is configured to face the reflecting surface of the beam splitter 2, that is, the outgoing light of the incident spectral light source can directly incident on the reflecting surface of the beam splitter 2, and after being reflected by the beam splitter 2, enter the light path of the microscope objective 1, thereby illuminating the surface of the object being observed.

[0041] Without limitation, the incident spectral source may be a tunable semiconductor laser.

[0042] In one embodiment of the present invention, the incident spectral light source is a monochromator combined with a xenon lamp to form a monochromatic light source. The xenon lamp serves as a broadband light source, emitting light signals covering the ultraviolet to near-infrared bands with stable light intensity and a continuous spectrum. After the emitted light from the xenon lamp enters the monochromator, it is dispersed by the monochromator's dispersive element, such as a reflective grating. The dispersive element separates light signals of different wavelengths, and by adjusting the angle of the dispersive element, a monochromatic light of a specific wavelength can be output.

[0043] Furthermore, the slit width of the monochromator is adjustable, with an adjustment range of 0.1 mm to 2 mm. By adjusting the slit width, the spectral resolution of the output light can be changed. The smaller the slit width, the higher the spectral resolution, but the lower the output light intensity. Therefore, in practical applications, it is necessary to balance spectral resolution and light intensity according to the observation requirements.

[0044] Optionally, in another embodiment of the present invention, the incident spectral light source is an array-type monochromatic LED light source. This type of light source consists of multiple LEDs emitting different wavelengths, each LED corresponding to a specific wavelength. The wavelength interval can be set as needed; for example, a wavelength interval of 5 nm can cover the visible light region from 400 nm to 700 nm. By sequentially lighting different LEDs, illumination of different wavelengths can be achieved.

[0045] The illumination method of the incident spectral light source is not limited to coupling to the optical path of the microscope objective 1 via the beam splitter 2. In an optional embodiment of the present invention, the incident spectral light source directly illuminates the object under observation via an external optical system. The external optical system includes a collimating lens and a condenser lens. The collimating lens is used to convert the outgoing light from the incident spectral light source into parallel light, and the condenser lens is used to converge the parallel light onto the surface of the object under observation, thereby improving the utilization rate of the illumination light. This illumination method is suitable for scenarios where it is inconvenient to set up the beam splitter 2 in the optical path of the microscope objective 1. For example, when the space of the outgoing optical path of the microscope objective 1 is limited, the external direct illumination method can simplify the optical path structure. Furthermore, the external optical system is also equipped with an aperture to adjust the field of view of the illumination light to match the field of view of the microscope objective 1, avoiding waste of illumination light.

[0046] Furthermore, in another optional embodiment of the present invention, the incident spectral light source illuminates the object of observation in an inverted manner via an external optical system. The external optical system includes a collimating lens, a condenser lens, and a stage. The stage is used to place the object of observation and has light-transmitting properties, for example, using a glass slide with a thickness of 0.17 mm. This thickness of the slide reduces the scattering and absorption of the light signal. The emitted light from the incident spectral light source is collimated by the collimating lens and converged by the condenser lens, then passes through the stage and illuminates the side of the object of observation opposite to the microscope objective 1. The light signal passes through the object and enters the microscope objective 1. This illumination method is suitable for transparent or translucent objects of observation, such as biological slices and thin film materials, and can achieve transmission-type hyperspectral imaging to obtain the spectral characteristics inside the object of observation. Furthermore, the numerical aperture of the condenser lens is matched with the numerical aperture of the microscope objective 1 to ensure that the numerical aperture of the illumination light is not less than the numerical aperture of the microscope objective 1, thereby improving the resolution and contrast of the imaging.

[0047] The device includes a tube lens 3, which is used to perform secondary imaging of the intermediate image formed by the microscope objective lens 1, so as to form a clear real image on the image plane 4.

[0048] Preferably, the tube mirror 3 is an infinity-corrected tube mirror 3, which matches the infinity-corrected characteristics of the microscope objective 1. Correspondingly, the object-side focal point of the tube mirror 3 coincides with the image-side focal point of the microscope objective 1, so that the parallel light emitted from the microscope objective 1 can be converged by the tube mirror 3 to form an image with stable magnification.

[0049] Furthermore, the tube mirror 3 is positioned in the optical path of the beam splitter 2 on the side opposite to the microscope objective 1, meaning that the light signal emitted from the microscope objective 1 and transmitted through the beam splitter 2 can be directly incident on the incident surface of the tube mirror 3 and transmitted to the image plane 4 after being converged by the tube mirror 3.

[0050] Furthermore, the focal length of the achromatic lens 3 is matched with the magnification of the microscope objective 1 to ensure that the final magnification of the image meets the requirements. Preferably, the achromatic lens 3 is an achromatic lens 3, which can correct chromatic aberration of light signals during transmission, ensuring that light signals of different wavelengths can form an image at the same position on the image plane 4 after passing through the lens 3, avoiding image blurring or shifting caused by chromatic aberration. Furthermore, the lens group of the achromatic lens 3 is made of two optical glasses with different dispersion characteristics, and the chromatic aberration is corrected through the structural design of the lens group.

[0051] In a preferred embodiment of the present invention, the aperture of the tube mirror 3 is not less than the diameter of the exit spot of the microscope objective 1 to avoid truncation of the light signal and ensure the field of view of the imaging. For example, if the diameter of the exit spot of the microscope objective 1 is 10 mm, then the aperture of the tube mirror 3 must be not less than 10 mm to ensure that all imaging light signals can enter the tube mirror 3. Furthermore, the surface of the tube mirror 3 is coated with an anti-reflection film to improve the transmittance of the light signal, reduce light loss, and thereby improve the light response intensity of the image plane 4.

[0052] The image plane 4 is an image acquisition component, used to receive the light signal after imaging by the tube lens 3 and convert the light signal into a digital image signal. Preferably, the image plane 4 is a CCD or CMOS image sensor. Furthermore, the image plane 4 is positioned at the image-side focal point of the tube lens 3, meaning that the real image formed by the tube lens 3 can be clearly presented on the photosensitive area of ​​the image plane 4, ensuring image clarity and integrity.

[0053] In one embodiment of the present invention, the image plane 4 also integrates a signal processing circuit. This signal processing circuit converts the optical signal received by the sensor into a digital electrical signal and performs preliminary noise reduction and amplification processing on the digital electrical signal to improve the quality of the image data. For example, the signal processing circuit employs correlated double sampling technology, which can effectively reduce the readout noise of the sensor; at the same time, it employs a programmable gain amplifier, which can adjust the amplification factor according to the intensity of the optical signal to avoid signal saturation or underexposure. Furthermore, the image plane 4 is connected to the image center processing system through a data transmission interface. Non-limitingly, the data transmission interface can be a USB 3.0 interface, a GigE interface, or a CameraLink interface, etc., wherein the GigE interface has a transmission speed of not less than 1Gbps, enabling high-speed data transmission, ensuring image acquisition efficiency, and is suitable for scenarios with high acquisition speed requirements.

[0054] The object of observation is positioned within the object-side working area of ​​the microscope objective 1, and the distance between the object and the object-side end face of the microscope objective 1 is the working distance of the microscope objective 1. The exit light path of the microscope objective 1 extends in a straight line, and the beam splitter 2 is positioned on this straight light path, with the light-transmitting surface of the beam splitter 2 forming a 45-degree angle with the straight light path. The exit light path of the incident spectral light source extends in a direction perpendicular to the exit light path of the microscope objective 1, and its exit light is incident on the reflecting surface of the beam splitter 2. The tube mirror 3 is positioned on the side of the beam splitter 2 away from the microscope objective 1, and the light-incident surface of the tube mirror 3 is coaxial with the exit light path of the microscope objective 1. The image plane 4 is positioned on the side of the tube mirror 3 away from the beam splitter 2, and the photosensitive area of ​​the image plane 4 coincides with the image-side focal point of the tube mirror 3.

[0055] Schematic illustration: The working distance between the object of observation and the microscope objective 1 can be determined according to the model of the microscope objective 1. For example, the working distance of a 10x microscope objective 1 is typically 4mm to 10mm, and the working distance of a 40x microscope objective 1 is typically 0.5mm to 2mm. During installation, it is necessary to ensure that the object of observation is within this working distance range to avoid collision between the microscope objective 1 and the object of observation, while ensuring clear imaging. Furthermore, the device is equipped with a focusing mechanism, which is used to adjust the distance between the microscope objective 1 and the object of observation to achieve clear imaging. The focusing mechanism can be a manual focusing knob or an electric focusing platform.

[0056] The distance between the beam splitter 2 and the microscope objective 1 needs to be determined based on the image-side working distance of the microscope objective 1. That is, the beam splitter 2 must be positioned outside the image-side working distance of the microscope objective 1 to ensure that the microscope objective 1 can form an image normally. For example, if the image-side working distance of the microscope objective 1 is 20mm, then the beam splitter 2 needs to be positioned at least 20mm away from the exit face of the microscope objective 1 to avoid affecting the imaging performance of the microscope objective 1. Furthermore, the beam splitter 2 is fixed on an adjustable bracket, which can adjust the angle and position of the beam splitter 2 to ensure that it forms a 45-degree angle with the exit light path of the microscope objective 1 and is on the correct optical path.

[0057] The distance between the tube mirror 3 and the beam splitter 2 is determined based on the object-side focal length of the tube mirror 3. Correspondingly, the object-side focal point of the tube mirror 3 coincides with the image-side focal point of the microscope objective 1. Therefore, the distance between the tube mirror 3 and the beam splitter 2 is the object-side focal length of the tube mirror 3 minus the distance between the beam splitter 2 and the image-side focal point of the microscope objective 1. Furthermore, the tube mirror 3 is fixed on a guide rail of the optical platform. The guide rail can adjust the position of the tube mirror 3 to achieve precise adjustment of the distance between it and the beam splitter 2.

[0058] The distance between the image plane 4 and the tube lens 3 is equal to the image-side focal length of the tube lens 3, ensuring that the real image formed by the tube lens 3 can be clearly presented on the photosensitive area of ​​the image plane 4. Furthermore, the image plane 4 is fixed on an adjustable slide, which can adjust the position of the image plane 4 to achieve precise adjustment of the distance between it and the tube lens 3, and simultaneously adjust the angle of the image plane 4 to ensure that the photosensitive area of ​​the image plane 4 is perpendicular to the optical axis of the tube lens 3, avoiding imaging distortion.

[0059] In practical applications, the microscope objective 1, beam splitter 2, tube mirror 3 and image plane 4 are fixed in the same optical platform or inside the microscope tube to avoid positional shifts caused by platform deformation; while the incident spectral light source is fixed on a support that is relatively stable relative to the beam splitter 2 to ensure that its outgoing light can be accurately incident on the reflecting surface of the beam splitter 2.

[0060] The hyperspectral microscope provided by this invention operates as follows: First, place the object to be observed on the stage in the object-side working area of ​​the microscope objective 1, and adjust the position of the stage so that the object to be observed is within the field of view of the microscope objective 1.

[0061] Then, the incident spectral light source is activated, causing it to emit monochromatic light of the first wavelength according to the preset wavelength sequence. This monochromatic light is incident on the reflective surface of the beam splitter 2 along its outgoing light path. After being reflected by the beam splitter 2, it enters the light path of the microscope objective 1 and is transmitted along the optical axis of the microscope objective 1, eventually illuminating the surface of the object being observed.

[0062] The surface of the object being observed reflects the single-spectrum light beam. If the object is opaque, it will be transmitted; if the object is transparent, the reflected or transmitted light signal carries the morphology and spectral information of the object and enters the object-side end face of the microscope objective 1. After being magnified by the lens group of the microscope objective 1, it forms parallel light and exits from the image-side end face of the microscope objective 1.

[0063] The parallel light propagates along the original optical path and is incident perpendicularly on the light-transmitting surface of the beam splitter 2. After being transmitted through the beam splitter 2, it enters the light-receiving surface of the tube mirror 3. The lens group of the tube mirror 3 converges the parallel light, forming a real microscopic image of the observed object at the first wavelength at the image-side focal point of the tube mirror 3. The photosensitive area of ​​the image plane 4 is located at the image-side focal point of the tube mirror 3, so it can receive the light signal corresponding to the real image. The sensor of the image plane 4 converts the light signal into an electrical signal. After noise reduction and amplification by the integrated signal processing circuit, it is converted into a digital image signal. Subsequently, the image plane 4 transmits the digital image signal to the image center processing system through the data transmission interface. The image center processing system stores and performs preliminary processing on the signal.

[0064] After acquiring the image at the first wavelength, the incident spectral light source switches to the second wavelength in the preset wavelength sequence, emitting monochromatic light of the second wavelength. The illumination, imaging, signal conversion, and transmission processes described above are repeated to acquire the digital image signal of the observed object at the second wavelength. Similarly, the incident spectral light source emits monochromatic light of different wavelengths sequentially according to the preset wavelength sequence, coordinating with the acquisition rhythm of image plane 4 to complete the acquisition of a preset number of digital image signals at different wavelengths. For example, if the preset wavelength sequence covers the band from 400nm to 700nm, with a wavelength interval of 5nm, then a total of 61 digital image signals at different wavelengths need to be acquired, with each wavelength corresponding to one microscopic image.

[0065] The aforementioned microscopic images at multiple wavelengths collectively constitute an image data cube. This data cube has two spatial dimensions corresponding to the x-axis and y-axis pixels of image plane 4, and one wavelength dimension corresponding to the wavelength of the incident spectral source. For example, if the pixel resolution of image plane 4 is 2048 x 2048, then the dimensions of the image data cube are 2048 x 2048 x 61. After receiving the image data cube, the image center processing system processes the data. Specifically, for each spatial pixel in the image data cube—that is, a pixel defined by the x-axis and y-axis—its grayscale values ​​at all wavelengths are extracted to form a spectral curve corresponding to that pixel. The horizontal axis of this spectral curve represents wavelength, and the vertical axis represents grayscale value, reflecting the spectral response characteristics at the corresponding location of the observed object.

[0066] Furthermore, the image center processing system can also perform subsequent processing and analysis on the image data cube, including, without limitation, the following steps: First, data denoising is performed using Gaussian filtering or median filtering algorithms to remove random noise from the image data and improve the signal-to-noise ratio. Second, spectral correction is performed by using the spectral curves of standard reference samples to correct the acquired pixel spectral curves, eliminating systematic errors and improving the accuracy of the spectral curves. Third, spectral matching is performed by comparing the corrected pixel spectral curves with those in the standard spectral library, and the composition at the corresponding location of the observed object is determined by calculating the correlation coefficient. Finally, visualization is performed by converting the pixel composition information into a pseudo-color image, where different components are associated with different colors to visually display the compositional distribution of the observed object.

[0067] In one embodiment of the present invention, the hyperspectral microscope is further equipped with an automatic control module, which is electrically connected to the incident spectral light source, the image plane 4, the stage and the focusing mechanism, for realizing automated hyperspectral image acquisition.

[0068] Specifically, the automatic control module operates as follows: First, it receives observation parameters input by the user, including wavelength range, wavelength interval, magnification, and acquisition field of view. Then, it controls the incident spectral light source to set the corresponding wavelength sequence according to the observation parameters. Subsequently, it controls the focusing mechanism to adjust the distance between the microscope objective 1 and the object being observed to achieve clear imaging. Next, it controls the incident spectral light source to emit monochromatic light sequentially according to the wavelength sequence, and triggers the image plane 4 to acquire data after each wavelength stabilizes, while simultaneously recording the acquired image data. If multiple regions of the object need to be acquired, it controls the stage to move, sequentially bringing different regions of the object into the field of view of the microscope objective 1, repeating the above illumination and acquisition steps to complete the acquisition of hyperspectral image data for multiple regions. After acquisition, it controls the incident spectral light source to turn off and transmits all acquired image data to the image center processing system for further processing.

[0069] Optionally, in another embodiment of the present invention, the hyperspectral microscope further includes a light shield, which is disposed on the outer side of the object end face of the microscope objective 1 and around the stage, for blocking ambient light from entering the imaging optical path and avoiding interference of ambient light with the imaging optical signal.

[0070] Preferably, the hyperspectral microscope further includes a calibration module, which is used to periodically calibrate the optical path and spectral accuracy of the device to ensure the accuracy of the acquired data.

[0071] The calibration module includes a standard reference sample and a calibration control unit. The standard reference sample has known spectral characteristics, such as a standard reflector or a standard transmission filter. The calibration control unit is electrically connected to the automatic control module and can control the movement of the stage to bring the standard reference sample into the field of view of the microscope objective 1. Then, it controls the incident spectral light source to emit monochromatic light according to a preset wavelength sequence to complete the hyperspectral image acquisition of the standard reference sample. The image center processing system compares the acquired spectral curve of the standard reference sample with the known standard spectral curve, calculates the spectral deviation of the system, and generates calibration parameters. The automatic control module corrects the wavelength output of the incident spectral light source and the signal acquisition of the image plane 4 according to the calibration parameters, thereby eliminating system errors. Furthermore, the calibration module can also calibrate the coaxiality of the optical path by acquiring images of the standard reference sample at different positions, analyzing the image offset, and controlling the adjustable support to adjust the positions of the beam splitter 2, the tube mirror 3, and the image plane 4.

[0072] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0073] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A hyperspectral microscope, characterized in that, The device includes a microscope objective (1), a beam splitter (2), a tube mirror (3), and an image plane (4). The microscope objective (1) is used to magnify and image the observed object and collect light signals. The beam splitter (2) is located in the image-side optical path of the microscope objective (1). The tube mirror (3) is located in the optical path of the beam splitter (2) on the side away from the microscope objective (1). The image plane (4) is located at the image-side focal point of the tube mirror (3) and is used to receive light signals and convert them into digital image signals.

2. The hyperspectral microscope according to claim 1, characterized in that, The microscope objective (1) is an infinity-corrected apochromatic objective whose spectral response range covers the working wavelength range of the incident spectral light source.

3. The hyperspectral microscope according to claim 1, characterized in that, The beam splitter (2) is a semi-transparent and semi-reflective beam splitter prism or a transparent thin sheet coated with a beam splitter film. Its light-transmitting surface is set at a preset angle to the outgoing light path of the microscope objective (1), so that the outgoing light of the microscope objective (1) is incident perpendicularly and transmitted along the original light path extension direction, while the outgoing light of the incident spectral light source is reflected into the light path of the microscope objective (1).

4. The hyperspectral microscope according to claim 3, characterized in that, The beam splitter (2) is a cubic beam splitter prism.

5. The hyperspectral microscope according to claim 1, characterized in that, It also includes an incident spectral light source, the outgoing light path of which is directed toward the reflective surface of the beam splitter (2), or directly irradiates the object of observation through an external optical system, or irradiates the object of observation inverted manner through an external optical system.

6. The hyperspectral microscope according to claim 5, characterized in that, The incident spectral light source is a monochromatic light source formed by a tunable laser, a monochromator, and a broadband light source, or an array of monochromatic LED light sources; the slit width of the monochromator is adjustable to balance spectral resolution and light intensity; the array of monochromatic LED light sources consists of multiple LEDs emitting different wavelengths, and different wavelengths of illumination are achieved by sequentially lighting different LEDs.

7. The hyperspectral microscope according to claim 1, characterized in that, The tube (3) is an infinity-corrected achromatic tube with its object-side focal point coinciding with the image-side focal point of the microscope objective (1). Its focal length matches the magnification of the microscope objective (1), which can correct the chromatic aberration during the transmission of light signals. Its aperture is adapted to the size of the emitted light spot of the microscope objective (1).

8. The hyperspectral microscope according to claim 1, characterized in that, The image plane (4) is a CCD or CMOS image sensor, which integrates a signal processing circuit for noise reduction and amplification of electrical signals, and is connected to the image center processing system through a data transmission interface.

9. The hyperspectral microscope according to claim 1, characterized in that, It also includes an automatic control module and a calibration module. The automatic control module is electrically connected to the incident spectral light source, the image plane (4), the stage and the focusing mechanism. The calibration module includes a standard reference sample and a calibration control unit, which are used to calibrate the optical path and spectral accuracy of the device.

10. The hyperspectral microscope according to claim 1, characterized in that, It also includes a light shield and a focusing mechanism. The light shield is located on the outer side of the object side of the microscope objective (1) and around the stage. The focusing mechanism is used to adjust the distance between the microscope objective (1) and the object being observed.

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