Transmission electron microscope in-situ sample rod with internal coaxial light path and test method

Abstract

The invention discloses a transmission electron microscope in-situ sample rod with an internal coaxial light path, and belongs to the field of transmission electron microscopes. The sample rod comprises a sample rod head, a sample rod body and a handle which are connected in sequence. The sample rod head is provided with a sample carrying table and a condensing lens, and the exciting light is converged to a sample; a sealed optical fiber guide channel and an optical fiber contained in the sealed optical fiber guide channel are arranged in the sample rod body, and the exciting light and fluorescence generated by exciting the sample are coaxially propagated through the optical fiber; a light beam collimator, a dichroscope, a light filtering assembly, a collecting lens and a sensor are arranged in the handheld handle, exciting light is collimated by the light beam collimator and reflected by the dichroscope, then is coupled to enter an optical fiber and is focused by a condensing lens to irradiate a sample, and a fluorescence signal generated by the sample is returned by the same optical fiber, is transmitted to pass through the dichroscope and the light filtering assembly and then is transmitted to the sensor. And the collecting lens focuses to the sensor. Through coaxial design of an excitation light path and a fluorescence light path, the internal space of the sample rod is remarkably saved, and in-situ and real-time monitoring of a material microscopic fluorescence dynamic process is realized.

Description

Technical Field

[0001] This invention relates to the field of transmission electron microscopy, and in particular to an in-situ optical sample holder for transmission electron microscopy with an internal coaxial optical path and a testing method thereof. Technical Background

[0002] Transmission electron microscopy (TEM) has become an important tool for materials characterization, and its importance in the field of materials science has been increasing in recent years. However, conventional TEM is limited by static observation and cannot capture the dynamic evolution of materials under real-world conditions. Therefore, in-situ TEM techniques have emerged. These techniques introduce external physical field excitations such as magnetism, light, force, heat, and electricity to achieve high spatiotemporal resolution dynamic real-time monitoring of the microscopic behavior of materials.

[0003] Optical properties are one of the key parameters for characterizing the performance of materials and devices, revealing various physical characteristics such as their electronic structure, composition, and energy conversion behavior. For example, Raman spectroscopy can reflect the lattice vibration modes and chemical bonding states of materials and is often used for structural characterization of carbon materials; while photoluminescence spectroscopy can be used to detect the defect state distribution and carrier recombination mechanism in perovskite semiconductors; when studying the effects of light irradiation on materials, it is usually necessary to irradiate the sample surface with light of a specific wavelength and intensity, and then analyze its dynamic changes such as photoresponse behavior, photocatalytic activity, or photodegradation process.

[0004] Transmission electron microscopy (TEM) provides high spatial resolution information on morphology, crystal structure, and composition, while fluorescence signals offer insights into band structure, defect states, exciton behavior, carrier dynamics, and other optical and electronic properties. Simultaneous acquisition of structural and optical information allows for the correlation of microstructural changes and optical responses within the same region and timescale, making it particularly suitable for: photoluminescence and phase transitions, and degradation processes in perovskite materials; and fluorescence quenching and energy transfer processes in quantum dots and carbon dots. Furthermore, many materials undergo photocatalysis, photodegradation, photoinduced phase transitions, enhanced luminescence, or quenching under light irradiation, which can be monitored in real-time through in-situ fluorescence collection.

[0005] Currently, conventional in-situ optical sample holders for transmission electron microscopes typically place the excitation and collection optical paths external to the sample holder. While this method simplifies the mechanical design, it has fundamental drawbacks: First, the externally fixed optical fiber cannot achieve precise and stable co-positioning with the sample position inside the electron microscope. The size and position of the light spot are prone to deviation and drift during long-path transmission, severely affecting the repeatability of experiments and the reliability of quantitative analysis. Second, due to the long and complex optical path, signal loss is significant, making it difficult to achieve efficient fluorescence collection, especially for weak signals or fast dynamic processes requiring time resolution.

[0006] Most importantly, the internal space of the transmission electron microscope sample rod (especially the part inserted into the microscope tube) is extremely limited. Under such stringent space constraints, it is almost impossible to accommodate two complete optical transmission systems if a traditional discrete optical path design (i.e., the excitation and collection optical paths are independent of each other) is adopted. This has become the main technical bottleneck for achieving high-performance optical detection inside the sample rod.

[0007] Therefore, a common technical bias exists in this field: integrating complex optical systems inside the sample holder of a transmission electron microscope is difficult and impractical. This leads researchers to prefer developing external optical path solutions, despite their inherent drawbacks. Overcoming this mindset, rationally arranging optical components within extremely limited space, and resolving a series of derivative problems such as optical path alignment, signal crosstalk, and vacuum maintenance have become pressing technical challenges in this field. Summary of the Invention

[0008] This invention addresses the shortcomings and defects of existing technologies by providing an in-situ transmission electron microscope (TEM) sample holder and testing method with an internal coaxial optical path. By coaxially integrating the excitation optical path and the fluorescence collection optical path within the sample holder, space is effectively saved and signal collection efficiency is improved. This invention is applicable to fluorescent materials, including but not limited to the following: perovskite materials, carbon dots, and quantum dot materials.

[0009] To achieve the above requirements, the present invention adopts the following technical solution:

[0010] The present invention provides an in-situ sample holder for transmission electron microscopy with an internal coaxial optical path, comprising a sample holder head, a sample holder body, and a handle connected in sequence.

[0011] The front end of the sample rod is provided with a sample stage for supporting the sample, and the rear end is provided with a condenser lens for focusing the excitation light;

[0012] The sample rod body has a sealed optical fiber guide channel running through it. The optical fiber guide channel contains an optical fiber, the front end of which extends to the rear end of the sample rod head and is coupled to the optical path of the condenser lens.

[0013] The handle includes a beam collimator, a dichroic mirror, a filter assembly, a collecting lens, and a sensor. The beam collimator is horizontally positioned in an opening on one side of the handle to receive and collimate excitation light emitted from an external laser. The dichroic mirror is tilted within the output optical path of the beam collimator, with its optical center on the extension of the fiber axis. The dichroic mirror reflects the collimated excitation light into the fiber and transmits the fluorescence signal returning from the fiber, meaning the excitation and fluorescence paths propagate coaxially through the dichroic mirror and the same fiber. The filter assembly is positioned within the fluorescence signal transmission path of the dichroic mirror to filter out residual excitation light from the fluorescence signal. The collecting lens focuses the fluorescence signal onto the sensor's photosensitive surface. The sensor is positioned at the focal point of the collecting lens to convert the fluorescence signal into an electrical signal and transmit it to an external signal processing device.

[0014] Preferably, to fix the sample, the sample stage is provided with a cover plate, and a first opening is provided at the intersection of the cover plate and the optical path to ensure the smooth flow of excitation light and fluorescence signal.

[0015] Preferably, the sample rod head and the sample rod body are detachably connected by a clamp.

[0016] Preferably, at least one sealing ring is provided on the outer side of the sample rod to maintain the vacuum environment of the transmission electron microscope tube.

[0017] Preferably, the optical fiber is a multimode optical fiber.

[0018] Preferably, the dichroic mirror is inclined at a 45° angle to the front end cap of the handle.

[0019] Preferably, the rear end cap of the handle is removable to facilitate the replacement of the corresponding dichroic mirror, filter assembly, and sensor as needed for testing.

[0020] Preferably, the condensing lens and the collecting lens are fixedly supported by a bracket with an integrated fine-tuning mechanism, which is used to precisely calibrate and fix the positions of the condensing lens and the collecting lens.

[0021] Preferably, the fine-tuning mechanism is a precision thread structure or a piezoelectric ceramic drive structure.

[0022] The present invention also provides a method for in-situ fluorescence testing using the sample rod, comprising the following steps:

[0023] Step 1: Place the sample on the sample stage and insert the sample rod into the transmission electron microscope so that the sample is in the electron beam path;

[0024] Step 2: Start the laser to generate excitation light of a specific wavelength. The excitation light is collimated into parallel light by the beam collimator and then propagates horizontally to the dichroic mirror. After being reflected by the mirror, the light changes direction and is coupled into the optical fiber in the optical fiber guide channel. The light is transmitted through the optical fiber to the rear end of the sample rod and then focused by the condenser lens to irradiate the sample.

[0025] Step 3: The sample is stimulated to generate a fluorescence signal. Part of the fluorescence signal returns along the original path, is collected by the focusing lens, coupled and transmitted back to the handle along the optical fiber, and is transmitted through the dichroic mirror. The filter component below the dichroic mirror filters out the residual excitation light in the fluorescence signal. The fluorescence signal of the target band is focused by the collecting lens to the sensor, which converts the fluorescence signal into an electrical signal.

[0026] Step 4: The sensor outputs the electrical signal to an external signal processing device to perform analysis and recording of fluorescence intensity spatiotemporal distribution, fluorescence lifetime measurement and imaging, fluorescence dynamics process, etc., and displays it on the display screen in real time.

[0027] Compared with the prior art, the embodiments of this application have at least the following beneficial effects:

[0028] (1) Since all core optical components (such as optical fibers, condenser lenses, dichroic mirrors, etc.) are rigidly integrated and precisely fixed inside the sample rod, a stable built-in optical path is formed. This avoids the problem of spot position drift caused by vibration, temperature fluctuation or insertion and removal operations of the external optical path, and ensures that the excitation spot and the sample position, as well as the fluorescence collection optical path, can achieve long-term, accurate and stable co-positioning, which greatly improves the repeatability and reliability of the experiment.

[0029] (2) By adopting a coaxial design for the excitation optical path and the fluorescence collection optical path, and using the same optical fiber for bidirectional transmission, the functions that traditionally require two independent optical path systems are integrated into a single channel. This fundamentally solves the structural contradiction between the extremely small internal space of the transmission electron microscope sample rod (especially the rod body) and the large size of the optical detection system, making it possible to integrate high-performance optical detection functions inside the sample rod.

[0030] (3) By highly integrating core functional modules such as beam collimator, dichroic mirror, filter, and sensor into the handle, and combining them with a detachable rear cover design, users can quickly and flexibly configure the entire optical system according to different samples and testing needs (such as changing lasers, dichroic mirrors, and filter groups of different wavelengths). This invention transforms a sample holder with a fixed function into a modular in-situ optical testing platform. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the in-situ sample holder for a transmission electron microscope with an internal coaxial optical path, as provided in an embodiment of this application.

[0032] Figure 2 A detailed schematic diagram of the in-situ sample rod head of the transmission electron microscope with an internal coaxial optical path provided in the embodiments of this application.

[0033] Figure 3 A schematic diagram of the internal structure of the transmission electron microscope in-situ sample rod and handle with an internal coaxial optical path provided in the embodiments of this application.

[0034] Figure 4 A top-view schematic diagram of the excitation optical path of the in-situ sample rod of a transmission electron microscope, which provides an embodiment of the present application for an internal coaxial optical path.

[0035] Figure 5 A top-view schematic diagram of the fluorescence optical path of the in-situ sample holder for a transmission electron microscope, provided as an embodiment of this application, showing the internal coaxial optical path.

[0036] In the picture:

[0037] 1-Sample rod head, 11-Sample stage, 12-Groove, 13-Condensing lens, 14-Cover plate, 15-Clamp, 111-First opening;

[0038] 2-Sample rod body, 21-Front end thin rod, 22-Rear end thick rod, 23-Fiber optic guide channel, 24-Fiber optic cable, 25-Sealing ring;

[0039] 3-Hand grip, 31-Guide sheath, 32-Beam collimator, 321-Second opening, 33-Dichroic mirror, 331-Emitting filter, 332-Bottom bracket, 34-Collecting lens, 35-CMOS sensor, 36-Interface, 37-Laser. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings. These embodiments are used to explain the present invention, but are not intended to limit its scope. It should be noted that the accompanying drawings are used to assist in explaining the structure and working principle of the present invention; their scale and dimensions may not necessarily reflect the actual size of the components. Some structures may be simplified, enlarged, or partially sectional for clarity, and the positions of certain components may be appropriately adjusted without affecting the implementation of the technical solution.

[0041] The directional descriptions used in this document, such as "front end," "rear end," "inner side," "outer side," "above," and "below," all refer to the end closer to the sample in the normal installation state as "front end" and the end farther from the sample as "rear end," and should be understood with reference to the conventional perspective shown in the accompanying drawings. They should not be construed as limiting the scope of protection of this invention.

[0042] Furthermore, the ordinal numbers such as "first" and "second" appearing in the instruction manual are only used to distinguish different parts with the same or similar structure, and do not indicate any order, quantity or importance.

[0043] The in-situ sample holder for transmission electron microscopes with an internal coaxial optical path provided by this invention achieves precise and real-time co-positioning with the sample position inside the electron microscope by integrating a precise fiber alignment and fixing structure inside the extremely narrow sample holder tip. This avoids deviations in the size and position of the light spot during transmission, and is particularly suitable for in-situ optical testing of fluorescent materials such as perovskite materials, carbon quantum dots, and quantum dot materials.

[0044] Figure 1 A schematic diagram of the overall structure of the in-situ sample rod is shown. In a specific embodiment of the present invention, the transmission electron microscope in-situ sample rod with an internal coaxial optical path mainly includes a sample rod head 1, a sample rod body 2, and a handle 3 connected in sequence.

[0045] like Figure 2 As shown, the sample rod head 1 has a plate-like structure with a sample stage 111 at the front end for supporting the sample. To fix the sample, a cover plate 14 is provided above the sample stage 11. The cover plate 14 has an opening for easy observation of the sample. At the same time, a first opening 111 is provided at the intersection of the cover plate 14 and the optical path to ensure the unobstructed flow of the excitation optical path and the fluorescence optical path. A rectangular groove 12 is formed in the middle of the sample rod head 1. A condenser lens 13 is fixed to the rear end of the groove 12 by an internal bracket. The condenser lens 13 is used to focus the excitation beam. The end of the sample rod body 1 is connected to the sample rod body 2 through a connector.

[0046] In one specific embodiment of the present invention, the bracket in the groove 12 is provided with a fine-tuning mechanism, such as a precision thread structure or a piezoelectric ceramic drive structure, for precisely calibrating and fixing the position of the condenser lens 13.

[0047] In one specific embodiment of the present invention, the sample rod head 1 and the sample rod body 2 are detachably connected by a clamp 15 at the end of the sample rod head 1.

[0048] Figure 3 The structure of the sample rod 2, the handle 3, and their internal optical components is shown. The sample rod 2 includes a thin front rod 21 with a smaller diameter and a thicker rear rod 22 with a larger diameter. A sealing device is provided on the outside of the interface between the two to maintain the ultra-high vacuum environment of the electron microscope tube and ensure the normal operation of the instrument. The thicker rear rod 22 is coaxially interference-fitted with the opening at the top of the handle 3. A sealed optical fiber guide channel 23 runs through the sample rod 2 to carry the optical fiber 24. The front end of the optical fiber 24 extends to the rear end of the sample rod head 1. The optical fiber 24, the condenser lens 13, and the sample are placed coaxially.

[0049] In one specific embodiment of the present invention, the sealing device on the outer side of the middle section of the sample rod 2 adopts at least one circular sealing ring 25.

[0050] In one specific embodiment of the present invention, optical fiber 24 is a multimode optical fiber with a large core diameter and a high numerical aperture, which can effectively collect more fluorescence signals.

[0051] like Figures 1 to 5 As shown, a guide sheath 31 is provided on the outer side of the front end cap of the handle 3, which cooperates with the guide groove of the transmission electron microscope sample stage interface to achieve precise insertion, removal and positioning of the sample rod; a second opening 321 is provided on one side of the front end of the handle, in which a beam collimator 32 is placed to ensure that the excitation light from the external laser 37 is horizontally incident; a dichroic mirror 33, a filter assembly, a collecting lens 34 and a sensor are arranged on the inner axis of the handle; the dichroic mirror 33 is a miniature optical element (diameter ≤10mm), which has high reflectivity to excitation light and high transmittance to fluorescence, and is used to reflect the excitation light emitted by the laser and transmit the fluorescence returned after the sample is excited. The dichroic mirror 33 is controlled by the hand An adjustable bracket is fixed inside the front cover of the handle, its optical center aligned with the center of the opening at the end of the rear thick rod 22 after calibration. The filter assembly and collecting lens 34 are both fixed by a bottom bracket 332 inside the handle. The filter assembly filters residual excitation light and allows the returned fluorescence to pass through. The fluorescence is focused by the collecting lens 34 and illuminates the photosensitive surface of the sensor. The sensor is positioned at the focal point of the collecting lens 34, receiving the fluorescence signal and converting it into an electrical signal. The sensor is preferably a CMOS sensor 35 or an sCMOS sensor, such as the Hamamatsu Orca-Flash 4.0 series or Teledyne Photometrics Prime series, which features high sensitivity, low noise, and fast response, suitable for detecting weak fluorescence signals. An interface 36 is provided on the rear cover of the handle to connect the sensor to external signal processing equipment and a display screen for real-time acquisition and analysis of fluorescence information. The rear cover of the handle 3 is removable, facilitating the replacement of the corresponding dichroic mirror 33 and filter assembly according to the sample. Simultaneously, the laser 37 can be replaced to match the absorption wavelength of the sample.

[0052] In one specific embodiment of the present invention, the installation angle of the selected dichroic mirror is preferably 45°. This angle needs to be precisely calibrated to ensure that the reflected light path is precisely aligned with the fiber axis. Any slight angular deviation will cause the excitation light to fail to couple effectively into the micron-sized fiber core, or cause the returned fluorescence signal to deviate significantly from the collection light path.

[0053] In one embodiment of the present invention, the filtering assembly is an emission filter 331 or a filter wheel composed of multiple filters. In another embodiment of the present invention, the bottom bracket 332 integrates a fine-tuning mechanism to precisely calibrate the position of the collecting lens 34, ensuring the coaxiality and focusing effect of the excitation light path and the fluorescence light path.

[0054] In one specific embodiment of the present invention, the external signal processing device connected to interface 36 may be selected as a computer data acquisition system, a time-correlated single-photon counter, a high-speed frame capture device, and other measurement devices that can be synchronized with the main system of the transmission electron microscope, as needed, to achieve in-situ correlation analysis under multi-physics coupling. By connecting different external devices, the present invention can extend the capabilities from static fluorescence observation to dynamic, time-resolved, and spectral-resolved multi-dimensional optical characterization.

[0055] When connected to a data acquisition card and a computer, the system operates in imaging mode. Two-dimensional fluorescence images acquired by the sensor are transmitted to the computer via the data acquisition card, and these images are correlated and registered with electron images acquired simultaneously by a transmission electron microscope. This allows for real-time observation of the fluorescence intensity distribution in microscopic regions such as perovskite grain boundaries and quantum dot aggregation areas, and the tracking of their dynamic evolution under electron beam irradiation or light illumination, generating images of the spatiotemporal distribution of fluorescence intensity.

[0056] When the laser is set to pulsed mode and connected to a time-correlated single-photon counter (TCSPC, such as Becker & Hickl's SPC series) + high-speed single-photon detector + computer, the sensor operates in single-photon counting mode. The TCSPC module records the arrival time of each fluorescence photon relative to the excitation laser pulse. By accumulating tens of thousands to millions of photons, a fluorescence lifetime decay curve is generated, and the fluorescence lifetime value can be obtained by fitting the curve. Furthermore, by scanning the sample or combining it with an area array detector, a fluorescence lifetime distribution map can be plotted for studying ultrafast processes such as exciton diffusion and energy transfer in materials.

[0057] When connected to a high sampling rate oscilloscope (such as the Tektronix DPO series) and a computer, the system can record the trajectory of fluorescence intensity changes over time in a specific micro-region, i.e., monitor fluorescence kinetics. This function can be used to capture the kinetic behavior of materials during phase transitions, degradation, or chemical reactions, such as fluorescence flickering, quenching, or enhancement.

[0058] This invention uses different optical elements to conduct experiments on different samples, and the following materials are used as examples:

[0059] Example 1

[0060] When testing perovskite materials, taking CH3NH3PbI3 as an example, its fluorescence peak is usually in the 700-800 nm range. At this time, laser 37 is a 532 nm solid-state laser; emission filter 331 is a 550 nm long-pass filter; and dichroic mirror 33 is a dichroic mirror with a reflection of 532 nm and a transmission of >550 nm.

[0061] Example 2

[0062] The carbon dot material was tested, and its fluorescence peak was in the range of 450-550 nm. At this time, the laser 37 was a 405 nm semiconductor laser; the emission filter 331 was a 450 nm long-pass filter; and the dichroic mirror 33 was a dichroic mirror with a reflection of 405 nm and a transmission of >450 nm.

[0063] Example 3

[0064] The quantum dot material was tested. Taking CdSe quantum dot as an example, its fluorescence peak is tunable and is commonly found in the range of 500-700 nm. At this time, the laser 37 is a 405 nm semiconductor laser; the emission filter 331 is a 500 nm long-pass filter; and the dichroic mirror 33 is a dichroic mirror with a reflection of 405 nm and a transmission of >500 nm.

[0065] This invention also provides a method for in-situ fluorescence testing using a transmission electron microscope in-situ sample holder based on the internal coaxial optical path, which is described below in conjunction with... Figure 4 excitation optical path and Figure 5 The fluorescence optical path description outlines the specific steps for in-situ fluorescence testing:

[0066] Step 1: Place the sample to be tested on the sample stage 11, insert the sample rod into the transmission electron microscope stage, and position the sample on the electron beam.

[0067] Step 2: As Figure 4 As shown, the laser 37 is activated to generate excitation light of a specific wavelength. The excitation light first enters the beam collimator 32 through the second opening 321 and is collimated into parallel light. The collimated beam propagates horizontally to the tilted dichroic mirror 33, and after being reflected by it, changes direction. It is then coupled into the optical fiber 24 located in the optical fiber guide channel 23 and transmitted to the rear end of the sample rod head 1. It is then focused by the condenser lens 13 and finally exits through the first opening 111 at the front end of the sample rod head 1, illuminating the sample to be tested fixed on the sample stage 11.

[0068] Step 3: As Figure 5 As shown, the fluorescence signal generated after the sample is excited is isotropic. Part of the fluorescence signal returns along the original path and is emitted through the opening 111 at the rear end of the sample rod head 1. It is collected by the condenser lens 13 and coupled into the optical fiber 24. The fluorescence signal is transmitted back into the handle 3 along the optical fiber 24 and passes through the dichroic mirror 33. It enters the filter assembly below it. After the filter assembly filters out the residual excitation light and other stray light, the fluorescence of the target wavelength band is focused by the collecting lens 34 and finally converges to illuminate the photosensitive surface of the sensor located at the bottom of the handle 3. The sensor converts the fluorescence signal into an electrical signal.

[0069] Step 4: The sensor outputs the converted electrical signal to an external signal processing device through interface 36, which can then perform fluorescence intensity spatiotemporal distribution imaging. When fluorescence lifetime measurement and imaging are required, the laser can be set to pulse mode and the sensor can be connected to a time-correlated single-photon counter, a high-speed single-photon detector, and a computer to perform fluorescence lifetime measurement and imaging. This invention can also connect the sensor to a high sampling rate oscilloscope and a computer to monitor the fluorescence dynamics process and display it on the screen in real time.

[0070] Through the above-mentioned coaxial optical path design, the present invention highly integrates excitation and signal collection functions within a limited sample rod space, thereby achieving efficient and real-time monitoring of the in-situ, dynamic optical processes of fluorescent materials in transmission electron microscopy.

[0071] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A transmission electron microscope in-situ sample holder with an internal coaxial optical path, characterized in that, It includes a sample rod head (1), a sample rod body (2), and a hand grip (3) connected in sequence. The front end of the sample rod is provided with a sample stage (11) for carrying the sample, and the rear end is provided with a condenser lens (13) for focusing the excitation light; The sample rod body (2) has a sealed optical fiber guide channel (23) inside, and the optical fiber guide channel contains an optical fiber (24). The front end of the optical fiber extends to the rear end of the sample rod head and is coupled to the optical path of the condenser lens. The handle includes a beam collimator (32), a dichroic mirror (33), a filter assembly, a collecting lens (34), and a CMOS sensor (35). The beam collimator is horizontally positioned in an opening on one side of the handle to receive and collimate the excitation light emitted from an external laser (37). The dichroic mirror is tilted in the output optical path of the beam collimator, with its optical center on the extension line of the fiber axis. The dichroic mirror reflects the collimated excitation light into the fiber and transmits the fluorescence signal returning from the fiber. That is, the excitation optical path and the fluorescence optical path are coaxially propagated through the dichroic mirror and the same fiber. The filter assembly is positioned in the fluorescence signal transmission optical path of the dichroic mirror to filter out residual excitation light in the fluorescence signal. The collecting lens is used to converge the fluorescence signal to the photosensitive surface of the CMOS sensor. The CMOS sensor is positioned at the focal point of the collecting lens to convert the fluorescence signal into an electrical signal and transmit it to an external signal processing device.

2. The transmission electron microscope in-situ sample holder according to claim 1, characterized in that, To fix the sample, a cover plate (14) is provided on the sample stage, and a first opening (111) is provided at the intersection of the cover plate and the optical path to ensure the smooth flow of excitation light and fluorescence signal.

3. The transmission electron microscope in-situ sample holder according to claim 1, characterized in that, The sample rod head and the sample rod body can be fixed together by a clamp.

4. The transmission electron microscope in-situ sample holder according to claim 1, characterized in that, The outer side of the sample rod is provided with at least one sealing ring (25) to maintain the vacuum environment of the transmission electron microscope tube.

5. The transmission electron microscope in-situ sample holder according to claim 1, characterized in that, The optical fiber is a multimode optical fiber.

6. The transmission electron microscope in-situ sample holder according to claim 1, characterized in that, The dichroic mirror is tilted at a 45° angle to the front cover of the handle.

7. The transmission electron microscope in-situ sample holder according to claim 1, characterized in that, The rear cover of the handle is removable, allowing for the replacement of the corresponding dichroic mirror, filter assembly, and CMOS sensor as needed for testing.

8. The transmission electron microscope in-situ sample holder according to claim 1, characterized in that, The condensing lens and the collecting lens are fixedly supported by a bracket with an integrated fine-tuning mechanism, which is used to precisely calibrate and fix the positions of the condensing lens and the collecting lens.

9. The transmission electron microscope in-situ sample holder according to claim 8, characterized in that, The fine-tuning mechanism is a precision thread structure or a piezoelectric ceramic drive structure.

10. A method for in-situ fluorescence testing using the sample rod as described in any one of claims 1-9, characterized in that, Includes the following steps: Step 1: Place the sample on the sample stage and insert the sample rod into the transmission electron microscope so that the sample is in the electron beam path; Step 2: Start the laser to generate excitation light of a specific wavelength. The excitation light is collimated into parallel light by the beam collimator and then propagates horizontally to the dichroic mirror. After being reflected by the mirror, the light changes direction and is coupled into the optical fiber in the optical fiber guide channel. The light is transmitted through the optical fiber to the rear end of the sample rod and then focused by the condenser lens to irradiate the sample. Step 3: The sample is stimulated to generate a fluorescence signal. Part of the fluorescence signal returns along the original path, is collected by the condenser lens, coupled and transmitted back to the handle along the optical fiber, and is transmitted through the dichroic mirror. The filter component below the dichroic mirror filters out the residual excitation light in the fluorescence signal. The fluorescence signal in the target band is focused by the collecting lens to the CMOS sensor, which converts the fluorescence signal into an electrical signal. Step 4: The sensor outputs the electrical signal to an external signal processing device, which then performs spatiotemporal distribution imaging of fluorescence intensity. Fluorescence lifetime measurement and imaging were performed by setting the laser to pulse mode and connecting the sensor to a time-correlated single-photon counter, a high-speed single-photon detector, and a computer. Connect the sensor to a high sampling rate oscilloscope and a computer to monitor the fluorescence dynamics process and display it on the screen in real time.

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