Portable in-situ self-scanning multispectral microscopic spectrum system

Abstract

The invention relates to a portable in-situ self-scanning multispectral microscopic spectrum system. The system comprises a multispectral microscopic spectrum measurement module, a movable self-supporting structure, a microscopic objective lens assembly and an objective lens scanning mechanism which are integrated; wherein the multispectral microscopic spectrum measurement module is used for realizing excitation and signal acquisition of multiple spectrum modes, and the spectrum modes comprise but are not limited to fluorescence spectrum, Raman spectrum and white light absorption spectrum; the microscope objective assembly is used as a unified in-situ optical interface, so that measurement of different spectrum functions is completed in the same light path and the same space coordinate system; the objective lens scanning mechanism is used for driving the microscope objective lens assembly to perform scanning movement in at least two space directions, so that multispectral space scanning measurement is realized under the condition that a measured object is kept relatively static, and state disturbance and measurement errors caused by moving a sample through an objective table traditionally are avoided; and the movable self-supporting structure is used for quickly deploying and keeping the system stable in different use scenes.

Description

Technical Field

[0001] This invention relates to the field of spectral detection and microscopic measurement technology, and in particular to a portable in-situ self-scanning multispectral microscopic spectral system. Background Technology

[0002] Spectroscopic detection techniques are one of the most commonly used and important methods in materials analysis and property characterization. By measuring samples using fluorescence spectroscopy, Raman scattering spectroscopy, white light absorption spectroscopy, multi-wavelength excitation spectroscopy, and time-resolved fluorescence spectroscopy, information such as the material's composition, energy level structure, defect characteristics, and luminescence dynamics can be obtained. Therefore, these spectroscopic techniques have been widely applied in materials science, semiconductor testing, biomedicine, and industrial testing.

[0003] Existing spectral detection systems mostly employ fixed or semi-fixed structures. When performing spatial scanning or multi-point measurements, spectral acquisition at different locations is typically achieved by moving the stage or sample platform, meaning the sample being measured is often in motion. Furthermore, in applications involving multiple spectral measurements, different spectral functions often need to be performed separately under different optical path configurations or equipment conditions. This makes it difficult to ensure the consistency of the same object's state across measurement processes in practical use, and the overall testing process is quite cumbersome.

[0004] Especially for samples that are sensitive to environmental conditions or mechanical disturbances, even minute displacements or changes in the state of the sample can affect the measurement results during scanning or repeated measurements. In addition, existing high-performance multispectral microscopy systems are usually large in size and highly dependent on the experimental environment, making them inconvenient to use in the field or in complex environments; while existing portable spectrometers generally suffer from limited spatial resolution and difficulty in achieving in-situ multispectral measurements at the microscale.

[0005] Currently, most spectral detection systems on the market are dedicated devices for single spectral techniques, such as standalone micro Raman spectrometers or micro fluorescence spectrometers. When multiple spectral characterizations of the same sample are required, it is usually necessary to transfer the sample between different devices or change different excitation sources and detection modules on the same device. This operating mode has the following significant drawbacks: First, the transfer of the sample between different devices or the switching between different optical paths on the same device makes it difficult to ensure accurate reproduction of the measurement points, resulting in different spectral data originating from different locations on the sample, which brings difficulties or even errors to data analysis; Second, traditional high-performance multispectral microscopy systems are usually complex in structure and large in size, requiring installation in an optical laboratory with a shockproof platform, which cannot meet the needs of on-site, in-situ, and rapid detection; Third, when performing spatial scanning imaging, existing technologies generally use a moving stage to change the measurement point, which will disturb the sample (especially liquid samples, live biological samples, or vibration-sensitive samples), change its original state, and introduce measurement errors.

[0006] To address the aforementioned issues, while some improvements have been made in existing technologies, such as coupling multiple light sources with optical fibers or using multi-band dichroic mirrors for beam combining, it remains difficult to simultaneously solve multiple technical challenges within a compact system, including multispectral functional integration, in-situ spatial consistency, sample disturbance-free scanning, and rapid on-site deployment. For example, while some solutions achieve multi-source beam combining, their optical path designs are complex, manual intervention may still be required when switching between different spectral modes, and the scanning method still relies on stage movement. Other solutions attempt to adopt lightweight designs, but often sacrifice spectral and spatial resolution, making it difficult to meet the precise measurement requirements at the microscopic scale.

[0007] Therefore, how to provide a multispectral microscopy system that can overcome the above-mentioned defects and has the advantages of multifunctional integration, high spatial consistency, in-situ scanning measurement and high portability is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a portable in-situ self-scanning multispectral microscopy system. This system enables integrated microscopic measurements using multiple spectral methods while keeping the object under test relatively stationary, and is suitable for rapid deployment in various application scenarios. Through innovative optical path design and modular integration, this system achieves in-situ scanning measurements of multiple spectral functions within the same microscopic optical path and spatial coordinate system while maintaining the absolute stationary state of the object under test. It also possesses rapid deployment capabilities to adapt to diverse field application scenarios.

[0009] The above-mentioned objective of this invention is achieved through the following technical solutions: This invention provides a portable in-situ self-scanning multispectral microscopy system, comprising: multiple excitation sources for providing excitation light with different wavelengths or time characteristics; optical isolators corresponding to the multiple excitation sources for suppressing the return of the excitation light; multiple mirrors for guiding and adjusting the propagation direction of the excitation light; a beam splitter for wavelength selection or beam splitting of white light excitation light; a beam combiner for combining excitation light from different excitation sources into the same excitation light path; a beam splitter A for guiding the combined excitation light to a microscopic excitation and imaging system; a microscopic excitation and imaging system for introducing the excitation light into the microscope objective and achieving sample imaging; and an objective scanning device for... While keeping the incident direction of the excitation light unchanged, the microscope objective is driven to scan and move in at least two spatial directions; the microscope objective is used to focus the excitation light onto the sample placement area and collect the light signal emitted by the sample; the spectral detection system is used to perform spectral analysis on the light signal emitted by the sample; wherein, the excitation light from the multiple excitation sources is introduced into the beam combiner and combined into the same excitation light path after passing through the optical isolator assembly, the reflector and the beam splitter, and then enters the microscopic excitation and imaging system through the beam splitter A and the reflector; the objective scanning device is configured to achieve multispectral spatial scanning measurement by spatial scanning of the microscope objective while keeping the sample placement area relatively stationary.

[0010] In this invention, by combining multiple excitation light sources, a single main optical path is ensured that excitation light of different spectral modes shares the same excitation light source. Combined with an objective scanning device, scanning measurements at different locations on the sample are achieved without moving the sample, thus avoiding sample disturbance. Simultaneously, the microscopic excitation and imaging system allows for real-time observation of the sample morphology and excitation spot position during spectral measurements, ensuring the accuracy of the measurement points.

[0011] According to one embodiment of the present invention, the multiple excitation light sources include: a first laser, which is a continuous narrow-linewidth laser, used for Raman spectroscopy measurements; a second laser, which is a frequency-tunable pulsed laser, used for single-wavelength excitation and time-resolved spectral measurements; and a third laser, which is a white-light continuous-spectrum laser, used for reflection absorption spectroscopy measurements or variable-wavelength excitation spectroscopy measurements. This light source configuration covers the mainstream spectral measurement needs from steady-state spectroscopy to transient dynamics, and from single-wavelength excitation to broadband absorption.

[0012] According to one embodiment of the present invention, the excitation light emitted by the first laser is sequentially introduced into a low-wavenumber device via a reflector before entering the beam combiner, so as to perform narrowband filtering on the laser line before participating in the beam combining. This low-wavenumber device can effectively filter out the sideband or amplified spontaneous emission (ASE) noise of the laser itself, and obtain pure laser with an ultra-narrow linewidth, which is crucial for realizing low-wavenumber Raman measurements and can detect molecular vibration information close to Rayleigh lines.

[0013] According to one embodiment of the present invention, the beam splitter is disposed in the optical path between the third laser and the beam combiner, and is used to select the wavelength of the white light excitation light, so that the third laser can be used as both a white light source and a variable wavelength excitation source. Preferably, the beam splitter is configured to switch between a white light continuous spectrum output mode and a monochromatic output mode for use in reflection absorption spectroscopy measurement and photoluminescence excitation spectroscopy (PLE) measurement, respectively. This design greatly enriches the functionality of a single light source.

[0014] According to one embodiment of the present invention, the objective scanning device includes: a first unidirectional moving stage on which a first small reflector is disposed and cooperates with a second small reflector to realize scanning of the excitation light in a first direction; a second unidirectional moving stage on which a third and a fourth small reflector are disposed for realizing scanning of the excitation light in a second direction perpendicular to the first direction; wherein the scanning directions of the first and second unidirectional moving stages are orthogonal to each other, thereby realizing two-dimensional scanning. This scanning structure composed of a moving stage and a fixed reflector pair can ensure that the outgoing beam is always parallel to the incident optical axis while changing the beam direction, which is the key to realizing objective lens normal incidence scanning.

[0015] According to one embodiment of the present invention, the microscope objective, the third small mirror, and the fourth small mirror are mounted in the same integrated scanning structure, so that the excitation light remains normally incident on the microscope objective during the scanning process. This scanning structure, consisting of a moving stage and a fixed mirror pair, can ensure that the outgoing beam is always parallel to the incident optical axis while changing the beam direction, which is the key to achieving normal incident scanning of the objective.

[0016] According to one embodiment of the present invention, a spectrometer is further included, which is used to receive the sample light signal collected by the microscope objective and returned along the original excitation optical path, and to perform spectral decomposition or splitting of the sample light signal for output. This "non-destructive" signal recovery method makes maximum use of weak light signals.

[0017] According to one embodiment of the present invention, the spectrometer includes a first output channel connected to a charge-coupled device (CCD) camera for rapidly acquiring spectral information of a sample, including but not limited to fluorescence spectra, reflectance spectra, or absorption spectra. The CCD camera has the advantage of multi-channel parallel detection, making it suitable for rapidly acquiring full-spectrum information.

[0018] According to one embodiment of the present invention, the spectrometer further includes a second output channel. After beam collimation by a collimating lens, the second output channel is introduced into a beam splitter D to achieve secondary splitting of the sample optical signal. Through secondary splitting, multiple point detectors with different functions can be connected simultaneously to meet different measurement requirements.

[0019] According to one embodiment of the present invention, the first shunt channel of the beam splitter D is connected to a photomultiplier tube, which is used to perform single-point intensity detection of the sample optical signal to achieve photoexcitation spectroscopy or single-point scanning spectral measurement. The photomultiplier tube has the characteristics of high sensitivity and fast response, making it suitable for intensity acquisition during wavelength scanning or spatial scanning.

[0020] According to one embodiment of the present invention, the second shunt channel of the beam splitter D is connected to a single-photon detector, which is used to detect single-photon signals emitted by the sample and, in conjunction with a time-correlated single-photon counting method, to achieve time-resolved fluorescence spectroscopy or carrier dynamics process measurement. This provides the possibility for studying ultrafast processes.

[0021] According to one embodiment of the present invention, the microscopic excitation and imaging system includes a beam splitter B and a beam splitter C. The beam splitter B is used to introduce the combined excitation light into the microscope objective system, and the beam splitter C is used to introduce and lead the illumination light and sample imaging light into and out of the microscopic imaging channel, so that the excitation light path and the imaging light path are coaxially arranged at the microscope objective. This coaxial design simplifies the system structure and avoids the parallax problem caused by off-axis imaging.

[0022] According to one embodiment of the present invention, the microscopic excitation and imaging system further includes a reflector and an illumination source. The illumination source is a broadband illumination source, and the illumination light emitted by it is collimated by the reflector and introduced into the microscope objective system through a beam splitter C for illuminating the sample placement area.

[0023] According to one embodiment of the present invention, the sample imaging light formed by reflection or transmission from the sample placement area is extracted by the microscope objective and beam splitter C and then enters the imaging camera to acquire real-time information on the sample surface morphology and the specific position of the excitation light on the sample surface. This allows the user to precisely locate the region of interest for spectral acquisition.

[0024] According to one embodiment of the present invention, the sample imaging information acquired by the imaging camera is used to assist in determining the excitation position of the excitation light on the sample placement area, thereby achieving consistency between the excitation position and the imaging position during multispectral measurement. This ensures that different spectral modes measure the same micro-region.

[0025] According to one embodiment of the present invention, the bundle combiner and / or bundle splitter A, bundle splitter B, bundle splitter C, and bundle splitter D include, but are not limited to, dichroic mirrors, cubic prisms, parallel plate bundle splitters, thin-film bundle splitters, fiber bundle combiners, or multi-port fiber optic devices. The specific selection depends on the wavelength range and energy requirements.

[0026] According to one embodiment of the present invention, the reflector adopts a reflective film layer formed by a metal film, a dielectric film, or a combination thereof, according to the wavelength range and power requirements of the corresponding excitation light source, so as to achieve efficient reflection.

[0027] According to one embodiment of the present invention, the spectrometer is configured to switch between a white light continuous spectrum output mode and a monochromatic output mode for use in reflection absorption spectroscopy measurement and wavelength-variable excitation spectroscopy measurement, respectively.

[0028] According to one embodiment of the present invention, the system is provided with an encapsulation shell for accommodating various optical devices and functional modules. The encapsulation shell is made of a metal material, engineering plastic, or composite material. The metal material includes aluminum alloy or stainless steel, the engineering plastic includes polycarbonate or ABS, and the composite material includes carbon fiber composite material or glass fiber reinforced composite material, or any combination of the above materials. The encapsulation shell not only serves a protective function but also forms the basis for achieving portability.

[0029] According to one embodiment of the present invention, the bottom of the encapsulation shell is provided with multiple movable self-supporting structures. These movable self-supporting structures are telescopic, used to adjust the overall height of the system to adapt to uneven ground conditions in different usage scenarios, and to achieve self-support and stable placement of the system without the need for an additional support platform. This allows the system to be quickly deployed anywhere, such as in the field, factory workshops, or archaeological sites.

[0030] In summary, compared with the prior art, the present invention has at least one of the following beneficial technical effects: High integration and multifunctionality: By combining multiple excitation sources into the same optical path and configuring a flexibly switchable detection system, multiple measurements such as fluorescence spectroscopy, Raman spectroscopy, absorption / reflection spectroscopy, photoexcitation spectroscopy, and time-resolved fluorescence spectroscopy can be performed on the same device, which greatly improves the utilization rate and working efficiency of the equipment.

[0031] In-situ measurement and spatial consistency: All spectral measurements share the same microscope objective and the same optical path, ensuring that different spectral data originate from the same micro-region of the sample, thus guaranteeing the comparability between data and the accuracy of correlation analysis.

[0032] Disturbance-free scanning measurement: A unique objective scanning device replaces the traditional stage movement, enabling spatial scanning while the sample remains stationary. This is particularly important for liquid samples, fragile samples, living samples, or samples in special environments (such as variable temperature or magnetic field), avoiding changes in state and measurement errors caused by sample movement.

[0033] High precision and high sensitivity: By combining low wavenumber devices and a variety of high-performance detectors (CCD, PMT, SPD), it meets the high-precision measurement requirements from conventional spectra to weak signals and from steady-state to transient dynamics.

[0034] Portability and environmental adaptability: Through compact optical path design, modular packaging and mobile self-supporting structure, the system is freed from dependence on fixed laboratories and can be quickly deployed and operate stably in complex and uneven field environments, greatly expanding the application scope of high-end spectroscopy technology. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the optical path of the excitation and beam combining module in an embodiment of the present invention.

[0036] Figure 2 This is a schematic diagram of the optical path of the imaging and illumination module in an embodiment of the present invention.

[0037] Figure 3 This is a schematic diagram of the objective lens scanning and in-situ testing module in an embodiment of the present invention.

[0038] Figure 4 This is a schematic diagram of the optical path of the spectral detection and splitting module in an embodiment of the present invention.

[0039] Figure 5 This is a schematic diagram of the encapsulated and movable self-supporting module in an embodiment of the present invention.

[0040] Figure 6 This is a schematic diagram of the module composition of the whole system in an embodiment of the present invention.

[0041] Reference numerals: 1. First laser; 2. Second laser; 3. Third laser; 4. Optical isolator A; 5. Reflector A; 6. Optical isolator B; 7. Reflector B; 8. Reflector C; 9. Beam splitter; 10. Beam combiner; 11. Beam splitter A; 12. Reflector D; 13. Low wavenumber device; 14. Reflector E; 15. Reflector F; 16. Reflector G; 17. Reflector H; 18. Beam splitter B; 19. Beam splitter C; 20. Reflector I; 21. Illumination source; 22. Imaging camera; 23. First small reflector; 24. Second small reflector; 25. First scanning direction; 26. First unidirectional moving stage; 27. Third small mirror; 28. Fourth small mirror; 29. ​​Second scanning direction; 30. Second unidirectional moving stage; 31. Microscope objective; 32. Sample placement area; 33. Spectrometer; 34. Collimating lens; 35. Beam splitter D; 36. Charge-coupled device camera (CCD); 37. Photomultiplier tube; 38. Single-photon detector; 39. Encapsulation shell; 40. First movable self-supporting structure; 41. Second movable self-supporting structure; 42. Third movable self-supporting structure; 43. Fourth movable self-supporting structure. Detailed Implementation

[0042] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0043] In the description of this application, it should be noted that the terms "upper," "lower," "inner," "outer," "top / bottom," etc., indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, and 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, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0044] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installed," "equipped with," "sleeved / connected," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0045] The portable in-situ self-scanning multispectral microscopy system provided by this invention includes an integrated multispectral microscopy measurement module, a movable self-supporting structure, a microscope objective assembly, and an objective scanning mechanism. The multispectral microscopy measurement module is used to achieve excitation and signal acquisition using various spectral methods, including but not limited to fluorescence spectroscopy, Raman spectroscopy, white light absorption spectroscopy, multi-wavelength excitation spectroscopy, and time-resolved fluorescence spectroscopy. The microscope objective assembly serves as a unified in-situ optical interface, used to complete excitation and signal collection under different spectral methods, enabling multiple spectral measurements to be performed within the same optical path and the same spatial coordinate system.

[0046] The objective scanning mechanism drives the microscope objective assembly to scan in at least two spatial directions, enabling multispectral spatial scanning measurements while the object under test remains relatively stationary, avoiding state changes or measurement errors caused by sample movement. A movable, self-supporting structure allows for rapid deployment in different application scenarios while maintaining overall stability during the measurement process.

[0047] This invention provides a portable in-situ self-scanning multispectral microscopy system that can perform various spectral functions, including but not limited to fluorescence spectroscopy, Raman spectroscopy, white light reflection / absorption spectroscopy, multi-wavelength excitation spectroscopy, and time-resolved fluorescence spectroscopy or carrier dynamics process measurement, under the same microscope objective 31 and the same spatial coordinate system. It can also achieve two-dimensional spatial scanning measurement under the condition that the sample is kept relatively stationary through the objective scanning device. At the same time, the system is equipped with a package shell 39 and movable self-supporting structures (including a first movable self-supporting structure 40, a second movable self-supporting structure 41, a third movable self-supporting structure 42, and a fourth movable self-supporting structure 43) to adapt to rapid deployment and stable placement in different scenarios.

[0048] For ease of explanation, the structure and function of this invention can be divided according to the accompanying drawings into an excitation and beam combining module, an imaging and illumination module, an objective lens scanning and in-situ testing module, a spectral detection and current splitting module, an encapsulation and self-supporting module, and a complete system module, as shown below. Figures 1 to 6 As shown.

[0049] like Figure 1As shown, in one embodiment, the excitation and beam combining module of the present invention is used to provide multiple excitation beams and combine different excitation beams into the same excitation optical path before introducing them into a microscopic excitation and imaging system. Its structure includes multiple excitation light sources, optical isolator components (including optical isolator A4 and optical isolator B6), mirrors (including mirrors A5, B7, C8, D12, E14, F15, G16, H17, and I20), a beam splitter 9, a beam combining device 10, a beam splitter A11, and a low wavenumber device 13.

[0050] Multiple excitation sources include: a first laser 1, preferably a continuous narrow linewidth laser, for Raman spectroscopy measurement; a second laser 2, preferably a frequency-tunable pulsed laser, for single-wavelength excitation and time-resolved measurement; and a third laser 3, preferably a white light continuous spectrum laser, for white light reflection / absorption measurement or variable wavelength excitation measurement obtained by the spectrometer 9.

[0051] The excitation light emitted by the first laser 1 is preferably first suppressed by optical isolator A4, then introduced into low-wavenumber device 13 for narrowband filtering via reflector A5 and reflector D12, before entering beam combiner 10 for beam combining. Low-wavenumber device 13 can be composed of gratings, filters, or combinations thereof, used to form an ultra-narrow laser line filtering channel to facilitate low-wavenumber Raman measurements. The optical isolator A4 is used here to improve beam command and prevent backscattering, but it is not mandatory.

[0052] The excitation light emitted by the second laser 2 is preferably suppressed from returning by the optical isolator B6, and then guided by the reflector B7 into the beam combiner 10 to participate in beam combining. The optical isolator B6 is used here to better control the beam and prevent backscattering, but it is not necessary.

[0053] The beam emitted by the third laser 3 preferably first enters the beam splitter 9 for wavelength selection or splitting; the beam splitter 9 includes, but is not limited to, one or more of AOTF, monochromator, beam splitting grating or prism, so that the third laser 3 can be in either white light continuous spectrum output mode or monochromatic output mode. The beam output by the beam splitter 9 is then guided into the subsequent optical path by the reflector C8 and the reflector E14.

[0054] The beam combiner 10 is used to combine beams from different excitation sources into the same excitation optical path. Its implementation includes, but is not limited to, one or more of the following: a dichroic mirror, a cubic prism, a parallel plate beam splitter, a thin-film beam splitter, an fiber optic combiner, or a multi-port fiber optic device. The combined excitation light is further guided by beam splitter A11 and reflectors F15, G16, and H17 into the region of the microscopic excitation and imaging system.

[0055] like Figure 2 As shown, in one embodiment, the imaging and illumination module of the present invention is used to realize the visualization observation of the sample area and the confirmation of the excitation point, and to make the excitation optical path and the imaging optical path coaxial at the microscope objective 31. Its structure includes a beam splitter B18, a beam splitter C19, a reflector I20, an illumination source 21, and an imaging camera 22.

[0056] The combined excitation light is introduced into the microscope objective 31 via beam splitter B18 to excite the sample. The illumination source 21 is a broadband illumination source, and its emitted illumination light is collimated by mirror I20 and then introduced into the microscope objective 31 via beam splitter C19 to illuminate the sample placement area 32. The sample imaging light is extracted from the microscope objective 31 and beam splitter C19 and enters the imaging camera 22 to acquire real-time information on the sample surface morphology and the specific position of the excitation light on the sample surface, thereby helping to determine the consistency between the excitation position and the imaging position during the multispectral measurement process.

[0057] In some embodiments, the excitation light introduced by the beam splitter B18 further enters the objective scanning and in-situ testing module, and is then guided by small mirrors (first small mirror 23, second small mirror 24, third small mirror 27, fourth small mirror 28) and unidirectional moving stages (first unidirectional moving stage 26, second unidirectional moving stage 30) to be orthogonally incident on the microscope objective 31.

[0058] like Figure 3 As shown, in one embodiment, the objective scanning and in-situ testing module of the present invention is used to realize two-dimensional scanning of the microscope objective 31 while keeping the sample placement area 32 relatively stationary. Its structure includes small mirrors (first small mirror 23, second small mirror 24, third small mirror 27, and fourth small mirror 28), a first unidirectional stage 26, a second unidirectional stage 30, the microscope objective 31, and the sample placement area 32.

[0059] The excitation light is preferably guided into the structure of the first unidirectional moving stage 26 by the first small reflector 23; the second small reflector 24 is disposed on the first unidirectional moving stage 26. By changing the relative position of the first small reflector 23 and the second small reflector 24, the excitation light is scanned in the first direction, while keeping the direction of the emitted beam unchanged or approximately unchanged.

[0060] Furthermore, the third miniature mirror 27 and the fourth miniature mirror 28 are disposed on the second unidirectional moving stage 30 and are orthogonal to the scanning direction of the first unidirectional moving stage 26, thereby realizing the scanning of the excitation light in the second direction and thus forming a two-dimensional scan. Preferably, the microscope objective 31 is mounted in the same integrated scanning structure as the third miniature mirror 27 and the fourth miniature mirror 28, so that the excitation light is always positively incident on the microscope objective 31 during the objective scanning process.

[0061] The sample placement area 32 adopts an open structure, which can be adapted to variable temperature, magnetic, pressurized or other in-situ environmental conditions.

[0062] like Figure 4 As shown, in one embodiment, the spectral detection and splitting module of the present invention is used to perform spectral analysis on the sample light signal collected by the microscope objective 31 and returned along the original excitation light path. Its structure includes a spectrometer 33, a collimating lens 34, a beam splitter D35, a charge-coupled device camera (CCD) 36, a photomultiplier tube 37, and a single-photon detector 38.

[0063] In some embodiments, the spectrometer 33 includes a first output channel and a second output channel: the first output channel is connected to a charge-coupled device camera (CCD) 36 to acquire the fluorescence spectrum, reflection spectrum, or absorption spectrum of the sample; the second output channel is collimated by a collimating lens 34 and then introduced into a beam splitter D35 for secondary splitting, wherein the first splitting channel of the beam splitter D35 is connected to a photomultiplier tube 37 for single-point intensity detection; the second splitting channel of the beam splitter D35 is connected to a single-photon detector 38 and, in conjunction with a time-correlated single-photon counting method, is used to realize time-resolved fluorescence spectroscopy or carrier dynamics process measurement.

[0064] like Figure 5 As shown, the present invention includes a packaging shell 39 for housing various optical devices and functional modules. The packaging shell 39 may be made of metal, engineering plastic, or composite material.

[0065] The bottom of the encapsulation shell 39 is provided with multiple movable self-supporting structures (first movable self-supporting structure 40, second movable self-supporting structure 41, third movable self-supporting structure 42, and fourth movable self-supporting structure 43). The movable self-supporting structures are telescopic structures used to adjust the overall height of the system to adapt to uneven ground conditions in different usage scenarios and to achieve self-support and stable placement of the system.

[0066] like Figure 6 As shown, Figure 6 A schematic diagram of the overall system of the present invention is shown, including an excitation and beam combining module, an imaging and illumination module, an objective scanning and in-situ testing module, a spectral detection and splitting module, and a packaging and movable self-supporting module. The present invention combines multiple excitation light sources (first laser 1, second laser 2, and third laser 3) into the same excitation optical path, and realizes a unified coordinate system for excitation, imaging, and signal recovery at the microscope objective 31. Combined with the objective scanning device, it achieves two-dimensional spatial scanning under conditions where the sample remains relatively stationary, thereby realizing multispectral microscopic scanning measurement under such conditions.

[0067] like Figure 6As shown, this embodiment provides a portable in-situ self-scanning multispectral microscopy system, which integrates multiple spectral measurement functions such as fluorescence spectroscopy, Raman spectroscopy, white light reflection / absorption spectroscopy, multi-wavelength excitation spectroscopy, and time-resolved fluorescence spectroscopy under the same microscope objective 31 and the same spatial coordinate system, and realizes two-dimensional spatial scanning measurement under the condition that the sample is kept relatively stationary through the objective scanning device.

[0068] In this embodiment, the system's excitation and beam combining module is as follows: Figure 1 As shown, the system is equipped with multiple excitation sources, including a first laser 1, a second laser 2, and a third laser 3. The first laser 1 is a continuous-wavelength narrow-linewidth laser, with preferred commonly used Raman test wavelengths including 405nm, 532nm, 785nm, and 1030nm in the long-wavelength region from visible light to near-infrared, used for steady-state fluorescence spectroscopy and Raman spectroscopy measurements. The second laser 2 is a frequency-tunable pulsed laser, with preferred commonly used excitation wavelengths including 405nm, 320nm, and 420nm in the shorter-wavelength region from visible light to ultraviolet, used for time-resolved fluorescence measurements. The third laser 3 is a white-light continuous-spectrum laser, mainly including halogen lamps, xenon lamps, and other gas ion sources, as well as continuous-spectrum white-light sources generated by photonic crystals, used for white-light reflection / absorption spectroscopy measurements or wavelength-selective wavelength-modulated excitation spectroscopy measurements.

[0069] When performing steady-state fluorescence or Raman spectroscopy measurements, the system selects a first laser 1 as the excitation source. The excitation light output from the first laser 1 is sequentially suppressed by optical isolator A4, and then guided by reflector A5 and reflector D12 into low-wavenumber device 13. Low-wavenumber device 13 is used to perform narrowband filtering on the laser line to improve the detection capability of low-wavenumber signals in Raman measurements. The excitation light processed by low-wavenumber device 13 enters beam combiner 10 and shares the same excitation optical path with other excitation sources.

[0070] When time-resolved fluorescence measurement is required, the system switches to the second laser 2 as the excitation source. The pulsed excitation light output from the second laser 2 is suppressed from returning by the optical isolator B6, and then guided by the reflector B7 into the beam combiner 10. It shares the subsequent optical path with the first laser 1, thus ensuring the consistency of the excitation position under different spectral measurement methods. Here, the beam combiner 10 is preferably a dichroic mirror; other options include beam splitters, multi-channel optical fibers, etc.

[0071] When white light reflection / absorption spectroscopy or multi-wavelength excitation spectroscopy measurements are required, the system selects a third laser 3 as the excitation source. The continuous white light spectrum output from the third laser 3 first enters the beam splitter 9, which can be an AOTF, monochromator, beam splitter grating, or prism, etc. Here, an AOTF is preferred for wavelength selection or beam splitting of the white light, which reduces the overall system size and makes the split beam relatively easy to collimate and tune. When the beam splitter 9 is in white light output mode, the system is used for reflection or absorption spectroscopy measurements; when the beam splitter 9 is in monochromatic output mode, the system is used for variable wavelength excitation spectroscopy measurements. The beam output from the beam splitter 9 is guided by mirrors (mirror C8, mirror E14) into the beam combiner 10.

[0072] Excitation beams from different excitation sources are combined into the same excitation beam path in the beam combiner 10, and then guided by the beam splitter A11 and the mirrors (mirror F15, mirror G16, mirror H17) into the microscopic excitation and imaging system.

[0073] Microscopic excitation and imaging systems such as Figure 2 As shown. The combined excitation light is introduced into the microscope objective 31 via beam splitter B18, forming a micro-excitation spot in the sample placement area 32. The illumination source 21 is a broadband illumination source. The illumination light emitted by it is collimated by mirror I20 and introduced into the microscope objective 31 via beam splitter C19 to illuminate the sample placement area 32. The imaging light formed on the sample surface is extracted by the microscope objective 31 and beam splitter C19 and enters the imaging camera 22 to observe the sample surface morphology and the specific position of the excitation light on the sample surface in real time, thereby assisting in determining the consistency between the excitation position and the imaging position in various spectral measurements.

[0074] After confirming the excitation position, the system enters the spectral measurement state. The fluorescence signal, Raman scattering signal, or reflection signal generated by the sample under the excitation light is returned along the original excitation light path via the microscope objective 31 and enters the spectral detection and splitting module, such as... Figure 4 As shown.

[0075] The sample light signal first enters the spectrometer 33. In this embodiment, the spectrometer 33 is equipped with a first output channel and a second output channel. The first output channel is connected to a charge-coupled device (CCD) camera 36 to quickly acquire information such as the fluorescence spectrum, reflection spectrum, or absorption spectrum of the sample. The second output channel, after being collimated by a collimating lens 34, is introduced into a beam splitter D35 for secondary splitting. The first splitting channel of the beam splitter D35 is connected to a photomultiplier tube 37 for single-point intensity detection to achieve photoexcitation spectroscopy or single-point scanning spectral measurement; the second splitting channel of the beam splitter D35 is connected to a single-photon detector 38 and, in conjunction with a time-correlated single-photon counting method, is used for time-resolved fluorescence spectroscopy or carrier dynamics process measurement.

[0076] During time-resolved fluorescence measurement, the system uses the second laser 2 as the excitation source while maintaining the microscopic excitation optical path unchanged. The electrical signal output by the single-photon detector 38 and the synchronization reference signal provided by the second laser 2 are input together into the time-correlated single-photon counting system to obtain time-resolved information on sample fluorescence decay.

[0077] In this embodiment, the system is also configured with an objective lens scanning and in-situ testing module, such as... Figure 3 As shown, before entering the microscope objective 31, the excitation light is guided sequentially by small mirrors (first small mirror 23, second small mirror 24, third small mirror 27, and fourth small mirror 28) and by the first unidirectional stage 26 and the second unidirectional stage 30. The excitation light is scanned in a first direction by driving the first unidirectional stage 26, and scanned in a second direction orthogonal to the first direction by driving the second unidirectional stage 30, thus achieving two-dimensional spatial scanning. Preferably, the microscope objective 31 and the small mirrors (third small mirror 27 and fourth small mirror 28) are mounted in the same integrated scanning structure, ensuring that the excitation light remains directly incident on the microscope objective 31 during the scanning process and that the sample remains relatively stationary.

[0078] In this embodiment, the unidirectional moving stage described is only a way of compressing the beam space. In actual use, it includes, but is not limited to, bidirectional moving stages, through mirrors, retroreflectors, optical microstructure designs, etc.

[0079] The sample placement area 32 adopts an open structure and can be configured with temperature-changing, magnetic, pressure-changing or other in-situ environmental devices as needed to meet the in-situ spectral measurement requirements under different experimental conditions.

[0080] In this embodiment, the entire system is encapsulated within the enclosure 39, such as... Figure 5 As shown. The enclosure 39 is used to house various optical components and functional modules, and its material can be aluminum alloy, stainless steel, engineering plastic, or composite material. The bottom of the enclosure 39 is provided with multiple movable self-supporting structures (first movable self-supporting structure 40, second movable self-supporting structure 41, third movable self-supporting structure 42, and fourth movable self-supporting structure 43). These movable self-supporting structures are telescopic, used to adjust the overall height of the system to adapt to uneven ground conditions in different usage scenarios, and to achieve self-support and stable placement of the system without the need for an additional support platform, thereby facilitating the transport and rapid deployment of the system between different testing scenarios.

[0081] As can be seen from the above embodiments, the portable in-situ self-scanning multispectral microscopy system provided by the present invention can achieve the integrated application of multiple spectral measurement methods under the same microscope objective and the same spatial coordinate system while keeping the sample relatively stationary. It can also complete two-dimensional spatial scanning measurement through the objective scanning device. It has the advantages of high integration of multiple functions, good spatial consistency, strong adaptability and easy on-site deployment. Example

[0082] This embodiment provides a portable in-situ self-scanning multispectral microscopy system, the overall structure of which is as follows: Figure 6 As shown, it mainly consists of an excitation and beam combining module ( Figure 1 Imaging and illumination module ( Figure 2 ), objective scanning and in-situ testing module ( Figure 3 ), spectral detection and splitting module ( Figure 4 ) and encapsulation and movable self-supporting modules ( Figure 5 This system consists of [components]. It enables high-spectral-resolution imaging in two-dimensional space through objective lens scanning without moving the sample, and allows for measurements of multiple spectral modes within the same micro-region.

[0083] I. Excitation and beam combining module (e.g.) Figure 1 (As shown) This module is used to generate and integrate multiple excitation beams, enabling them to propagate along the same main optical path.

[0084] The module includes multiple excitation light sources, specifically: First laser 1: In this embodiment, a continuous wave (CW) narrow-linewidth semiconductor laser with a center wavelength of 785nm is selected. Its linewidth is less than 0.1nm, and it is mainly used for Raman spectroscopy measurements. This wavelength can effectively avoid fluorescence background interference generated by most samples.

[0085] Second laser 2: In this embodiment, a frequency-tunable picosecond pulse laser with a wavelength of 405nm is selected, and the repetition frequency is adjustable (e.g., 1-80MHz) to realize time-resolved fluorescence spectroscopy measurement under single-wavelength excitation in order to study the lifetime dynamics of charge carriers.

[0086] Third laser 3: In this embodiment, a supercontinuum white laser is selected, with a spectral coverage range of 450-2400nm, for measuring white light reflection / absorption spectra. Simultaneously, in conjunction with the subsequent beam splitting device, it can also serve as a variable wavelength excitation source.

[0087] The optical paths of each light source are as follows: The laser emitted by the first laser 1 first passes through an optical isolator A4 (e.g., an optical isolator based on the Faraday effect) to prevent light reflected back from subsequent optical interfaces from interfering with or damaging the laser. After the emitted light is oriented by a reflector A5, it is introduced into a low-wavenumber device 13 through a reflector D12. In this embodiment, the low-wavenumber device 13 consists of a volume Bragg grating (VBG) or a high-precision Fabry-Perot interferometer. Its function is to further compress the laser linewidth and suppress the sidebands and background noise of the laser, thereby obtaining an ultra-pure single-mode excitation light, which is crucial for detecting low-wavenumber (e.g., <100 cm⁻¹) Raman signals. The beam, after narrowband filtering, is guided to a beam combiner 10.

[0088] The pulsed laser emitted by the second laser 2 is also first protected by the optical isolator B6, and then the optical path is directly changed by the reflector B7 and guided to the beam combiner 10.

[0089] The supercontinuum white light emitted by the third laser 3 first enters the beam splitter 9. In this embodiment, the beam splitter 9 is an AOTF (Optical Aperture Tolerant Fiber) beam splitter. AOTFs have advantages such as being fully electronic, having no moving parts, having a fast wavelength switching speed (on the order of microseconds), and having an adjustable passband. By controlling the frequency of the radio frequency signal applied to the AOTF, monochromatic light of any wavelength can be quickly and accurately selected from the broadband white light (the linewidth can be adjusted to a few nanometers), or all wavelengths of light can be selected to pass through (i.e., white light mode). The beam after being split by the AOTF may be monochromatic light or broadband light, depending on the mode, and is then guided to the beam combiner 10 by the reflector C8 and the reflector E14.

[0090] The beam combiner 10 is the core of this module. In this embodiment, it is a specially coated dichroic mirror assembly or a multi-channel fiber optic combiner. For example, if a dichroic mirror is used, the mirror needs to have high transmittance for the light from the first laser 1 (785nm) and the second laser 2 (405nm), while having high reflectivity for the light from the third laser 3 (e.g., tunable light in the 450-700nm range) after AOTF splitting, thereby achieving three-way beam combining. The combined excitation light then passes through beam splitter A11. In this embodiment, beam splitter A11 is a key component for separating the excitation light and the signal light, such as a planar beam splitter with flat sidebands or a dichroic mirror with a high damage threshold. After being reflected by beam splitter A11, the combined light is guided sequentially by reflector F15, reflector G16, and reflector H17, finally sending the beam into the imaging and illumination module.

[0091] II. Imaging and Illumination Module (e.g.) Figure 2 (As shown) This module is used to achieve white light illumination and real-time imaging of the sample, and to ensure that the excitation light and the imaging optical path are coaxial.

[0092] The combined excitation light from the excitation and beam combining module is first incident on beam splitter B18. The function of beam splitter B18 is to efficiently introduce the excitation light into the subsequent objective scanning system, while allowing the signal light returning from the sample to pass efficiently and enter the detection module. After being reflected by beam splitter B18, the excitation light enters the objective scanning and in-situ testing module.

[0093] The illumination source 21 is a broadband LED white light source. The light emitted from it is collimated and oriented by the reflector I20, then reflected by the beam splitter C19 and enters the objective scanning module along the same path as the excitation light, ultimately illuminating the sample. The beam splitter C19 is a broadband planar beam splitter with a 50 / 50 splitting ratio in the visible light band, which can efficiently reflect the illumination light towards the sample and efficiently transmit the imaging light returning from the sample to the imaging camera 22.

[0094] The imaging optical path is as follows: After the sample is illuminated by white light, its surface morphology information is collected by the microscope objective 31, returns along the original path, first passes through the objective scanning module, then sequentially passes through beam splitter B18 and beam splitter C19, and finally images onto the photosensitive surface of the imaging camera 22. The imaging camera 22 can be a high-resolution CMOS or CCD camera. Through the camera, the user can observe the microstructure of the sample surface in real time and precisely position the excitation spot to the region of interest by adjusting the system. A mark is usually superimposed on the camera image to indicate the position of the excitation spot, thus ensuring that what is seen is what is measured.

[0095] III. Objective lens scanning and in-situ testing module (e.g.) Figure 3 (As shown) This module is one of the core innovations of this invention, used to achieve two-dimensional scanning by moving the microscope objective without moving the sample.

[0096] The module mainly includes: a first small reflector 23, a second small reflector 24, a first one-way moving stage 26, a third small reflector 27, a fourth small reflector 28, a second one-way moving stage 30, and a microscope objective 31.

[0097] The excitation light from the imaging and illumination module first strikes a fixed first miniature mirror 23 and is reflected onto a second miniature mirror 24 mounted on a first unidirectional stage 26. The first unidirectional stage 26 can move precisely in a first scanning direction 25 (e.g., the X direction). As the first unidirectional stage 26 moves, the position of the light spot incident on the second miniature mirror 24 changes accordingly, but because the second miniature mirror 24 moves with the stage, the direction of its emitted light remains unchanged; only the beam undergoes a translation in a plane perpendicular to the optical axis. This translated beam is then guided to the next scanning stage by a fixed pair of mirrors (not shown in detail in the figure).

[0098] Next, the light beam is incident on a mirror pair consisting of a third miniature mirror 27 and a fourth miniature mirror 28 mounted on the second unidirectional moving stage 30. The second unidirectional moving stage 30 is placed on the first unidirectional moving stage 26, so when the first unidirectional moving stage moves, the second unidirectional moving stage 30 moves accordingly, but the first unidirectional moving stage 26 can remain unchanged when the second unidirectional moving stage 30 moves. The second unidirectional moving stage 30 can move precisely in a second scanning direction 29 (e.g., the Y direction) perpendicular to the first scanning direction. Its working principle is similar to that of the first stage: by moving the mirror pair (the third miniature mirror 27 and the fourth miniature mirror 28), the light beam is translated in the Y direction while keeping the direction of the emitted light unchanged.

[0099] After two stages of orthogonal translation, the beam is introduced into the microscope objective 31, which is rigidly connected to the third miniature mirror 27 and the fourth miniature mirror 28. Since the mirrors (third miniature mirror 27, fourth miniature mirror 28) and the microscope objective 31 are mounted as a single unit on the second unidirectional stage 30, the outgoing beam remains incident directly into the rear aperture of the microscope objective 31 regardless of the stage's combined movement in the X and Y directions. This ensures that the quality of the excitation spot focused on the sample placement area 32 remains highly consistent throughout the scanning process.

[0100] The sample placement area 32 is open and can integrate various in-situ sample environment accessories, such as variable temperature heating / cooling stages, electromagnets, stretching stages, liquid pools, or atmosphere control chambers. During objective scanning, the sample itself and its surrounding environment remain absolutely still, maximizing the protection of the sample's original state and the stability of the environment.

[0101] IV. Spectral Detection and Stream Splitting Module (e.g.) Figure 4 (As shown) This module is used to collect and analyze the light signals emitted by the sample.

[0102] The Raman scattering, fluorescence, and reflected / transmitted light signals generated after the sample is excited are collected by the microscope objective 31 and returned along the original excitation light path. The returned signal light passes sequentially through the objective scanning module, through the beam splitter B18, and is then guided into the entrance slit of the spectrometer 33.

[0103] In this embodiment, spectrometer 33 is an image-corrected spectrometer with a focal length of 300 mm, equipped with three automatically switchable gratings (e.g., 150 gr / mm for broadband fluorescence, and 600 gr / mm and 1200 gr / mm for high-resolution Raman). The spectrometer disperses the incident signal light according to wavelength, forming a spectral distribution at its exit focal plane. Spectrometer 33 is designed with two output ports: First output port: This port is directly connected to a high-sensitivity, deep-cooled CCD camera. When the system performs routine fluorescence spectroscopy, Raman spectroscopy, or white light reflectance / absorption spectroscopy measurements, the CCD camera can simultaneously acquire signals across the entire spectral range, offering the advantages of speed and intuitiveness.

[0104] Second output port: The light output from this port has already undergone dispersion, but the spectral image is extracted outside the spectrometer. The outgoing light is first collimated by a collimating lens 34, and then incident on a beam splitter D35. Beam splitter D35 splits the beam into two paths: The first shunt channel connects to the photomultiplier tube 37 (PMT). The PMT has extremely high response speed and single-photon level sensitivity. When the system performs photoluminescence excitation (PLE) spectroscopy measurements, the excitation wavelength of the third laser 3 needs to be simultaneously tuned (via the AOTF), and the intensity of the emission at a specific emission wavelength is recorded using the PMT. When the system performs mapping imaging based on objective scanning, the PMT can be used in conjunction with the fixed wavelength output of the monochromator to achieve intensity distribution imaging of the sample surface at a specific wavelength.

[0105] The second shunt channel is connected to a single-photon detector 38 (SPD), such as a single-photon counter based on an avalanche photodiode (APD). This channel is used when the system performs time-resolved fluorescence lifetime measurements. The output signal of the SPD and the synchronous trigger signal of the second laser 2 are input together into the time-correlated single-photon counting (TCSPC) module to obtain the decay curve of the sample fluorescence intensity, thereby calculating the fluorescence lifetime and analyzing the carrier dynamics.

[0106] V. Packaging and movable self-supporting modules (such as...) Figure 5 (As shown) This module is used to integrate and protect all the aforementioned optical and mechanical components, and to enable the system to be portable and deployed quickly.

[0107] All optical components, the stage, detectors, etc., are precisely mounted inside a rigid, lightweight enclosure 39. The enclosure 39 is made of carbon fiber composite material to ensure good thermal and mechanical stability while reducing weight. The enclosure is designed with dust protection for the optical path and electromagnetic shielding.

[0108] At the four bottom corners of the enclosure 39, first, second, third, and fourth movable self-supporting structures (first movable self-supporting structure 40, second movable self-supporting structure 41, third movable self-supporting structure 42, and fourth movable self-supporting structure 43) are respectively installed. In this embodiment, each self-supporting structure is a combination device integrating rollers and adjustable support feet. When the system needs to be moved, the system can be slightly lifted, the adjustable support feet can be rotated off the ground, and the rollers can be used for transport. After the system is transported to the designated location, the adjustable support feet are rotated to lower them to contact the ground and support the entire system, at which point the rollers are suspended in the air. By adjusting the height of the four support feet respectively, uneven ground can be compensated for, ensuring that the system obtains a stable and vibration-free measurement platform in any location, eliminating the need to find and transport an air-bearing optical platform.

[0109] VI. System Workflow Example Sample positioning and imaging: Place the sample in the sample placement area 32. Turn on the illumination source 21, observe the sample through the imaging camera 22, move the entire system or use the coarse positioning function of the objective scanning module to move the area of ​​interest to the center of the field of view and focus precisely.

[0110] Raman spectroscopy measurement: The first laser 1 is selected as the excitation source. Through software control, the excitation light is directed along the aforementioned optical path to illuminate the sample. The returned signal light is dispersed by the spectrometer 33 and then acquired by the CCD camera to obtain the Raman spectrum at that point. If Raman mapping is required, the objective scanning module is activated, controlling the first unidirectional stage 26 and the second unidirectional stage 30 to move according to preset step sizes and paths, acquiring a spectrum at each scanning point using the CCD.

[0111] Fluorescence spectroscopy and fluorescence lifetime measurement: Switch to the second laser 2 as the excitation source. Acquire the signal again while keeping the sample position unchanged. For steady-state fluorescence spectroscopy, CCD acquisition is still used; for fluorescence lifetime measurement, the signal from the second output port is guided to the single-photon detector 38 via beam splitter D35, and the TCSPC module is activated for data acquisition.

[0112] Absorption / Reflection Spectroscopy and PLE Spectroscopy Measurement: Switch to the third laser 3 and spectrometer 9. If the reflection / absorption spectrum needs to be measured, set the spectrometer 9 to white light mode, illuminate the sample with white light, and use spectrometer 33 and CCD to acquire the reference light and sample signals respectively, calculating the absorption / reflection spectrum. If the PLE spectrum needs to be measured, set the spectrometer 9 to scanning monochromatic light mode, use the PMT as the detector, and record the curve of sample luminescence intensity changing with excitation wavelength at a specific emission wavelength.

[0113] Throughout the entire process, the sample remains stationary. This procedure allows users to obtain complementary multispectral information within the same micro-region, providing comprehensive and accurate data support for a deeper understanding of sample characteristics. Example

[0114] This embodiment is basically the same as Embodiment 1, except for the selection of some components and the adjustment of the optical path.

[0115] Wavelength selection for the excitation source: Depending on the application requirements, the first laser 1 can be a 532nm or 633nm laser. For example, for resonant Raman measurements, a wavelength matching the sample's electronic absorption band can be selected. The second laser 2 can be a shorter wavelength ultraviolet laser (such as 266nm or 355nm) for exciting wide-bandgap semiconductor materials or for ultraviolet fluorescence studies. The third laser 3 can be a high-power xenon lamp with a filter wheel instead of a supercontinuum laser to reduce costs.

[0116] Selection of beam combiners and beam splitters: In Example 1, if an optical fiber beam combiner is used as the beam combiner 10, three laser beams can be coupled into a single-mode or multimode optical fiber. This ensures that the beams emitted from the fiber are naturally coaxial, making optical path adjustment simpler and improving the system's anti-interference capability. Correspondingly, beam splitters such as A11 also need to be selected based on the characteristics of the optical fiber's output light.

[0117] Variations of the objective scanning device: The first unidirectional moving stage 26 and the second unidirectional moving stage 30 can be replaced by piezoelectric ceramic-driven nano-positioning stages instead of traditional lead screw stepper motor stages to achieve higher precision and faster scanning speeds. Simultaneously, the mirrors (first miniature mirror 23, second miniature mirror 24, third miniature mirror 27, and fourth miniature mirror 28) can be designed as an integrated scanning head, for example, by coating the mirrors onto a single optical prism, further reducing size and improving stability.

[0118] Variations of the spectrometer 9: In addition to the AOTF, a liquid crystal tunable filter (LCTF) or a miniaturized grating monochromator can also be used. The LCTF also has no moving parts and provides good image quality; although the miniaturized monochromator is slightly slower in switching speed, it can provide higher spectral resolution.

[0119] Variations of the spectral detection system: In a low-cost version that only requires steady-state spectral measurements and not time-resolved and PLE measurements, the second output channel, beam splitter D35, PMT, and SPD can be omitted, retaining only the spectrometer 33 and CCD. Conversely, in a high-end version, independent preamplifiers and photon counters can be configured for the PMT and SPD to improve dynamic range and sensitivity.

[0120] Variations in packaging materials: The packaging shell 39 can also be made of aluminum alloy or stainless steel, precision machined to provide higher structural rigidity and heat dissipation performance, suitable for laboratory environments with extremely high stability requirements, while retaining its ability to be deployed in a mobile manner. Example

[0121] This embodiment focuses on the usage of the system in a specific application scenario, further highlighting its practicality.

[0122] Application Scenario 1: Archaeological Site Artifact Analysis The system was transported to the archaeological excavation site. Due to the potentially uneven ground, the system was kept level and stable by adjusting four movable self-supporting structures (first movable self-supporting structure 40, second movable self-supporting structure 41, third movable self-supporting structure 42, and fourth movable self-supporting structure 43). The ancient mural fragments or ceramic samples to be analyzed were placed in sample placement area 32. First, the microstructure and pigment particles of the sample were observed using white light illumination and imaging camera 22. Then, Raman spectroscopy analysis of the area of ​​interest was performed using the first laser 1 (e.g., 785 nm) to quickly identify the mineral composition of the pigments (e.g., cinnabar, lapis lazuli, etc.). Next, the system was switched to white light mode using the third laser 3 to measure the spectral reflectance of the area and obtain color information. Finally, if it was necessary to understand the organic binders or trace fluorescent substances in the pigments, the system could be switched to the second laser 2 (e.g., 405 nm) for fluorescence spectroscopy measurement. The entire process did not require moving the sample, and all spectral data came from the same micrometer-scale area, greatly improving the accuracy of correlation between different analytical results. The system can perform in-situ, non-destructive, multi-spectral analysis on-site, avoiding the risks of transporting precious cultural relic samples back to the laboratory.

[0123] Application Scenario 2: Semiconductor Wafer Defect Detection In the semiconductor manufacturing workshop, the system is placed next to the wafer transfer box. The system is leveled using support structures (first movable self-supporting structure 40, second movable self-supporting structure 41, third movable self-supporting structure 42, and fourth movable self-supporting structure 43). A wafer is placed in the open sample placement area 32. The imaging module quickly locates suspected defects (such as particles or scratches) on the wafer. Micro-Raman spectroscopy is performed on this point using the first laser 1, and the characteristic peaks are used to determine whether the defect is amorphous silicon, carbide, or other contaminants. To assess the impact of defects on luminescence performance, the system can switch to the second laser 2 for photoluminescence (PL) spectroscopy to obtain the luminescence intensity and peak position information of the defect area, determining whether it is a recombination center affecting device efficiency. The objective scanning module can perform small-scale Raman or PL mapping on the defect and its surrounding area, generating a two-dimensional distribution map of the defect's impact. All measurements are performed with the wafer stationary, avoiding positioning errors and potential contamination risks caused by moving large wafers.

[0124] Application Scenario 3: Dynamic Monitoring of Living Cells The system is placed in the clean bench of the cell culture chamber. A live cell sample in a culture dish is placed in sample placement area 32, and a CO2 and temperature control system is connected to maintain cell viability. The target cells are located using the imaging module (illumination source 21, imaging camera 22). First, a bright-field image of the cells is recorded using low-power white light (third laser 3, white light mode). Then, a specific wavelength of excitation light (e.g., 488 nm selected by the third laser 3 and the spectrometer 9) is used to excite a specific fluorescent protein (such as GFP) within the cells, and its fluorescence spectrum and intensity are acquired using a CCD to monitor protein expression. Subsequently, the system switches to pulsed light from the second laser 2 (e.g., 405 nm) and the detection channel is switched to SPD. The fluorescence lifetime of the fluorescent protein is measured using TCSPC technology, which is highly sensitive to environmental factors (such as pH and ion concentration), allowing for non-invasive monitoring of changes in the intracellular microenvironment. Since the sample stage remains stationary during objective scanning, the cells in the culture dish are not disturbed by shaking, enabling long-term, stable dynamic monitoring.

[0125] In summary, this invention, through its unique overall design, successfully integrates multiple advanced spectroscopic technologies onto a portable platform. Furthermore, through an innovative objective scanning scheme, it achieves in-situ, multimodal, and high-precision spectral imaging analysis of stationary samples, demonstrating significant technological advancements and broad industrial application prospects. This invention enables in-situ, multispectral, and scannable microscopic spectral detection in complex sites or non-fixed laboratory environments, combining advantages such as multifunctional integration, spatial consistency, convenient deployment, and measurement stability.

[0126] The implementation principle of this invention is as follows: This invention discloses a portable in-situ self-scanning multispectral microscopic spectral system, belonging to the field of spectral detection and microscopic measurement technology. The system includes multiple excitation light sources, a beam combiner 10, a microscopic excitation and imaging system, an objective scanning device, a microscope objective 31, and a spectral detection system. Multiple excitation beams are combined into the same optical path and then introduced into the microscope objective 31 via the microscopic excitation and imaging system. The objective scanning device drives the microscope objective 31 to scan in at least two directions while keeping the excitation light incident direction unchanged, achieving spatial scanning measurement while keeping the sample stationary. This invention integrates multiple spectral functions into the same optical path and employs objective scanning technology, enabling multispectral measurements such as fluorescence, Raman, and absorption, as well as two-dimensional spectral imaging of the same micro-region while keeping the sample in its in-situ condition, avoiding disturbances caused by sample movement. Simultaneously, the system features a movable self-supporting structure, facilitating rapid deployment in complex environments, and combining multifunctionality, high precision, and portability.

[0127] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A portable in-situ self-scanning multispectral microscopy system, characterized in that, include: Multiple excitation sources are available to provide excitation light with different wavelengths or time characteristics; Optical isolator components, each corresponding to one of the various excitation light sources, are used to suppress the return of excitation light; Multiple mirrors are used to guide and adjust the propagation direction of the excitation light; Spectrometer (9) is used for wavelength selection or spectral splitting of white light excitation light; A beam combiner (10) is used to combine excitation beams from different excitation sources into the same excitation optical path; Beam splitter A (11) is used to guide the combined excitation light to the micro-excitation and imaging system; A microscopic excitation and imaging system is used to introduce excitation light into the microscope objective (31) and to achieve sample imaging; An objective scanning device is used to drive a microscope objective to scan in at least two spatial directions while keeping the incident direction of the excitation light constant. A microscope objective (31) is used to focus the excitation light onto the sample placement area (32) and collect the light signal emitted by the sample; A spectral detection system is used to perform spectral analysis on the light signal emitted by the sample. The excitation light from the various excitation sources is introduced into the beam combiner (10) and combined into the same excitation light path after passing through the optical isolator assembly, the mirror and the beam splitter (9). Then it enters the microscopic excitation and imaging system through the beam splitter A (11) and the mirror. The objective scanning device is configured to perform multispectral spatial scanning measurements by spatial scanning of the microscope objective (31) while the sample placement area (32) remains relatively stationary.

2. The portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, Before entering the beam combiner (10), the excitation light emitted by the first laser (1) is sequentially introduced into the low wavenumber device (13) through a reflector to perform narrowband filtering on the laser line before participating in the beam combiner.

3. A portable in-situ self-scanning multispectral microscopy system according to claim 1 or 2, characterized in that, The beam splitting device (9) is located in the optical path between the third laser (3) and the beam combining device (10) to select the wavelength of the white light excitation light, so that the third laser (3) can be used as a white light source or as a variable wavelength excitation source.

4. The portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, The objective scanning device includes: The first unidirectional moving stage (26) is provided with a first small reflector (24) and cooperates with a second small reflector (23) to realize the scanning of the excitation light in the first direction; The second unidirectional moving stage (30) is provided with a third small reflector (27) and a fourth small reflector (28) for scanning the excitation light in a second direction perpendicular to the first direction; The second unidirectional moving stage (30) is mounted on the first unidirectional moving stage (26); the scanning directions of the first unidirectional moving stage (26) and the second unidirectional moving stage (30) are orthogonal to each other, thereby realizing two-dimensional scanning.

5. The portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, The microscope objective (31) is mounted in the same integrated scanning structure as the third small mirror (27) and the fourth small mirror (28) so that the excitation light is always positively incident on the microscope objective (31) during the scanning process.

6. The portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, The spectral detection system includes a spectrometer (33), which is used to receive the sample light signal collected by the microscope objective (31) and returned along the original excitation light path, and to perform spectral decomposition on the sample light signal; the spectrometer is provided with multiple output channels, wherein: the first output channel is connected to the charge-coupled device camera CCD (36) to acquire the fluorescence spectrum, reflection spectrum or absorption spectrum of the sample; the second output channel is connected to the photomultiplier tube (37) and the single photon detector (38) respectively after being split by the collimating lens (34) and the beam splitter D (35), and is used to realize single-point intensity detection and time-correlated single photon counting measurement.

7. The portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, The microscopic excitation and imaging system includes a beam splitter B (18) and a beam splitter C (19). The beam splitter B (18) is used to introduce the combined excitation light into the microscope objective system. The beam splitter C (19) is used to introduce and extract the illumination light and sample imaging light into the microscopic imaging channel, so that the excitation light path and the imaging light path are coaxially set at the microscope objective (31). The system also includes an illumination source (21) and a reflector (20). The illumination light emitted by the illumination source (21) is collimated by the reflector (20) and introduced into the microscope objective system through the beam splitter C (19) to illuminate the sample placement area (32). The sample imaging light formed by reflection or transmission from the sample placement area (32) is extracted by the microscope objective (31) and the beam splitter C (19) and enters the imaging camera (22) to obtain the surface morphology information of the sample and the position of the excitation light on the sample surface.

8. The portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, The sample imaging light formed by reflection or transmission from the sample placement area (32) is drawn out by the microscope objective (31) and beam splitter C (19) and enters the imaging camera (22) to obtain the sample surface morphology information and the specific position of the excitation light on the sample surface in real time.

9. A portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, The spectrometer (9) is configured to switch between a white light continuous spectrum output mode and a monochromatic output mode for use in reflection absorption spectroscopy measurement and wavelength-modulated excitation spectroscopy measurement, respectively.

10. A portable in-situ self-scanning multispectral microscopy system according to claim 1, characterized in that, The system is equipped with a housing (39) for accommodating various optical devices and functional modules. The housing (39) is made of metal, engineering plastic, or composite material. The metal material includes aluminum alloy or stainless steel, the engineering plastic includes polycarbonate or ABS, and the composite material includes carbon fiber composite or glass fiber reinforced composite, or any combination of the above materials. Multiple movable self-supporting structures are provided at the bottom of the housing (39). These movable self-supporting structures are telescopic and are used to adjust the overall height of the system to adapt to uneven ground conditions in different usage scenarios, and to achieve self-support and stable placement of the system without the need for an additional support platform.

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