Optical fiber micro-beam probe and system capable of realizing synchronous imaging of cell morphology and mechanical property

Abstract

The invention discloses an optical fiber micro-beam probe and system capable of achieving synchronous imaging of cell morphology and mechanical characteristics, the optical fiber micro-beam probe comprises a single-mode optical fiber, a Bragg grating, a supporting column, a micro-beam and a nanometer needle tip, the single-mode optical fiber comprises a fiber core and a wrapping layer outside the fiber core, and the Bragg grating is located in the fiber core of the single-mode optical fiber; the first end of the supporting column is fixed to the second end face of the single-mode optical fiber, one end of the micro-beam is fixed to the second end of the supporting column, and the nanometer needle tip is located on the surface of the other end of the micro-beam. The metal film is introduced to enhance the reflectivity of the micro-beam, and the nano needle tip is introduced to improve the cell morphology and mechanical imaging resolution. An optical fiber FPI sensitive external weak force is formed on the end face of an optical fiber by adopting a micro-beam probe, resonance of the micro-beam probe is excited by light driving, the resonance is influenced by the weak attractive force on the surface of a cell, the change of information such as resonant frequency and amplitude is monitored, and synchronous imaging of the morphology and the mechanical property of the living cell is realized. Miniaturization, integration and high efficiency of an imaging system are facilitated, and a new method is provided for cell imaging in the fields of biomedicine and the like.

Description

Technical Field

[0001] The present invention belongs to the field of fiber optic sensors, and relates to an all-fiber cell imaging system, in particular to a preparation method of an optical fiber microbeam probe based on the cavity optomechanical effect, and an all-fiber cell morphology and mechanical property synchronous imaging measurement system. Background Art

[0002] The morphological characteristics and mechanical properties of cells are two basic properties of cells, which can participate in regulating many physiological processes such as cell growth, differentiation, migration, and apoptosis. In addition, the morphology and mechanical properties of cells (such as Young's modulus and adhesion force, etc.) can reflect the potential of tumors to develop into malignancy, and are important properties in the basic research of tumor treatment. Therefore, achieving high spatio-temporal resolution imaging of the morphology and mechanical properties of living cells is of great significance for better understanding cell characteristics, exploring cell expression, revealing the mechanism of cell carcinogenesis, and further exploring new methods for cancer diagnosis and treatment.

[0003] Currently, the existing cell imaging technologies mainly include: optical microscopy imaging, photoacoustic imaging, fluorescence imaging, and atomic force microscope (AFM) imaging, etc. These imaging technologies can obtain cell morphology or mechanical properties, but they all have their own technical limitations, such as being unable to simultaneously obtain the morphology and mechanical information of living cells during imaging, and having problems such as large volume, complex operation, low resolution, and difficulty in synchronous measurement of multiple parameters. Therefore, the existing technologies need to be improved.

[0004] The all-fiber cell imaging system has strong anti-electromagnetic interference ability, high stability, can realize synchronous imaging of cell morphology and mechanical properties, and at the same time has the advantages of small volume, flexible use, high resolution, and no need for labeling, which are incomparable to many traditional cell imaging systems.

[0005] CN106501551A discloses an optical fiber-based atomic force microscope probe and an atomic force microscope system. The optical fiber-based atomic force microscope probe includes a probe and a microcantilever beam. The probe is located at one end of the microcantilever beam, and the microcantilever beam is located at one end of the optical fiber. An optical fiber F-P cavity is formed between the microcantilever beam and one end face of the optical fiber through a connecting arm. The microcantilever beam is used to sense the change in the distance between the probe and the sample. The following problems exist in this patent: (1) In terms of function realization: Using the microcantilever beam to sense the van der Waals force on the sample surface can only achieve static measurement of the single morphology of ordinary samples, and cannot achieve synchronous imaging of cell morphology and mechanical properties. (2) In terms of working mode: This optical fiber-based atomic force microscope probe operates in a static mode, cannot achieve multi-parameter synchronous measurement, and has low sensitivity and resolution. (3) In terms of preparation: The optical fiber-based atomic force microscope probe is integrally processed by surface micromachining technology (picosecond laser processing, ultrasonic vibration-assisted processing) and bulk micromachining technology (dry etching, anisotropic wet etching). The processing steps are cumbersome, the processes are complex, the processing accuracy is low, and the materials of the microcantilever beam and the probe are single (only glass material), and it is impossible to prepare a probe with a size below 1 nm. Summary of the Invention

[0006] A fiber optic microbeam probe and system capable of synchronous imaging of cell morphology and mechanical properties proposed by the present invention uses an integrated fiber optic microbeam probe as a carrier, utilizes the cavity optomechanical effect of an optical fiber Fabry-Perot interferometer (FPI) cavity, and integrates all-optical driving and all-optical detection technologies to achieve simultaneous high spatio-temporal resolution imaging of the morphology and mechanical properties of living cells. The fiber optic microbeam probe FPI perfectly combines two advantages: namely, the high spatial resolution of an integrated nanoscale tip and the high temporal resolution of optically driven FPI. By using the driving light to excite multiple resonant frequencies, different resonant modes of the microbeam probe are excited. The signal of the first-order resonant mode is used for morphology imaging, and the signal of the second-order resonant mode is used for mechanical property imaging, realizing simultaneous recording of different characteristic information of cells and providing a new method for solving the simultaneous high spatio-temporal resolution imaging of the morphology and mechanical properties of living cells.

[0007] The present invention realizes cell imaging based on the cavity optomechanical effect of an optical fiber FPI. Its basic principle is: A modulated optical signal beam is incident into the FPI as the driving (excitation) light, thereby driving the mechanical resonance of the microbeam probe. When the nanoscale tip approaches a living cell and scans its surface, the mutual attractive force between the tip and the cell surface will affect the mechanical resonance state of the microbeam probe. By using another probe light beam to demodulate the changes in state information such as resonant frequency, amplitude, and phase, simultaneous high spatio-temporal resolution imaging of the morphology and mechanical properties of living cells is achieved.

[0008] The present invention aims to solve the above problems of the prior art, and the technical solutions adopted are as follows:

[0009] An optical fiber microbeam probe capable of synchronously imaging cell morphology and mechanical properties includes a single-mode optical fiber, a Bragg grating, a support column, a microbeam, and a nano tip. The single-mode optical fiber includes a core and a cladding outside the core, and has a first end face and a second end face. The Bragg grating is located in the core of the single-mode optical fiber. The first end of the support column is fixed to the second end face of the single-mode optical fiber. One end of the microbeam is fixed to the second end of the support column. The nano tip is located on the surface of the other end of the microbeam. The second end face of the single-mode optical fiber, the support column, the microbeam, and the nano tip together form an optical fiber Fabry–Pérot interferometer (FPI). An optical microcavity is formed between the second end face of the single-mode optical fiber and the microbeam. The single-mode optical fiber is used for the transmission of driving light and detection light. The optical microcavity can form an optical resonant cavity for exciting the cavity optomechanical effect. The surface of the microbeam can reflect optical signals for demodulation. At the same time, the microbeam probe, as a mechanical resonator, is sensitive to the weak attraction on the cell surface, thereby affecting the resonance of the microbeam probe. The fiber FPI can be used to provide interference light. By demodulating the changes in the state information such as the resonance frequency, amplitude, and phase of the microbeam probe with the detection light, synchronous imaging of cell morphology and mechanical properties based on the cavity optomechanical effect is achieved.

[0010] Furthermore, a metal thin film is plated on the microbeam and the nano tip. The metal thin film is a gold, silver, or copper metal thin film. The Bragg grating and the metal thin film are mutually matched to improve the contrast and fineness of the reflected optical signal. By changing the shape and size of the microbeam, the working frequency and working bandwidth of the optical fiber microbeam probe can be changed, and further the working bandwidth and time resolution of the cell imaging system can be changed.

[0011] Furthermore, the metal thin film is a gold, silver, or copper metal thin film.

[0012] Furthermore, the microbeam includes a microbeam substrate, a mass block, and a mirror. The microbeam substrate is a locally hollowed-out thin sheet structure, and at least one end is a triangular tip for fixing to the nano tip. The mass block is arranged in the hollow part of the microbeam substrate so that the mass block can move horizontally along the microbeam substrate. The mirror is arranged on the surface of the microbeam substrate. The horizontal movement of the mass block along the microbeam substrate can be achieved by setting sliding grooves on the microbeam substrate or at both ends of the mass block. The mirror can be circular and composed of a circular thin sheet and four support rods. The four support rods are used for the fixed connection between the circular thin sheet and the microbeam substrate. The circular thin sheet is located directly below the core of the single-mode optical fiber.

[0013] The present invention also provides a method for preparing an optical fiber microbeam probe capable of synchronously imaging cell morphology and mechanical properties, comprising the following steps: First, a Bragg grating is prepared in the core of a single-mode optical fiber; Second, support columns, a microbeam, and a common tip are prepared on the flattened end face of the single-mode optical fiber by using femtosecond laser two-photon polymerization technology; Third, the common tip in the previous step is etched (focused ion beam (FIB) etching or plasma etching) to realize the preparation of a nano tip; Finally, a metal thin film is prepared on the surfaces of the microbeam and the nano tip. Then, the processed single-mode optical fiber, Bragg grating, support columns, microbeam, and nano tip together constitute the optical fiber microbeam probe.

[0014] Further, the method for preparing the Bragg grating is to prepare the Bragg grating in the core by using femtosecond laser direct writing technology or the phase mask method.

[0015] Further, the method for preparing the support columns, microbeam, and common tip includes: First, a polymer photoresist solution is coated on the end face of the optical fiber, then the structural dimensions and shapes of the support columns, microbeam, and common tip are optimized by using finite element simulation, then the designed support columns, microbeam, and common tip structures are printed on the second end face of the optical fiber by using femtosecond laser two-photon polymerization technology, and finally the printed structures are developed by using photoresist development technology.

[0016] Further, the metal thin film is prepared on the surfaces of the microbeam and the nano tip by using magnetron sputtering coating or evaporation.

[0017] The present invention finally also provides a system capable of synchronously imaging cell morphology and mechanical properties, comprising an optical fiber microbeam probe, a driving light source, a detection light source, a signal generator, an electro-optic modulator, a three-dimensional piezoelectric nano scanner, a feedback controller, a computer, a circulator, a coupler, a tunable optical filter, a photodetector, and an analyzer (electrical spectrum analyzer or vector network analyzer or lock-in amplifier);

[0018] The driving light source emits driving light, and the signal generated by the signal generator modulates the driving light through the electro-optic modulator. The modulated driving light reaches the optical fiber microbeam probe through the coupler and the circulator, exciting the microbeam probe to generate resonance;

[0019] The detection light source emits a detection light signal, and this light signal successively reaches the optical fiber microbeam probe through the coupler and the circulator;

[0020] The three-dimensional piezoelectric nano scanner is arranged below the optical fiber microbeam probe, and a living cell or tissue is placed on the three-dimensional piezoelectric nano scanner. When the nano tip approaches the living cell or tissue and scans its surface, the mutual attractive force between the tip and the cell or tissue surface will affect the resonance state of the microbeam probe;

[0021] The probe light signal is reflected and transmitted at the second end face of the single-mode fiber, and then reflected again on the surface of the microbeam. The two reflected light beams form an interference light signal in the fiber, and then enter the analyzer through the circulator, tunable optical filter and photodetector, so as to obtain the first-order and second-order resonant states of the microbeam probe.

[0022] The analyzer is connected to the computer, and the feedback controller is respectively connected to the computer and the three-dimensional piezoelectric nanoscanner.

[0023] The analyzer detects the changes in amplitude and phase in the first-order resonant state, and controls the movement of the three-dimensional piezoelectric nanoscanner in the Z-axis direction through the feedback controller, so that its first-order amplitude remains at the initial set value, and records the movement trajectory of the three-dimensional piezoelectric nanoscanner, so as to realize the tracking of the microscopic morphology of the cell surface; by detecting the changes in frequency and phase in the second-order resonant state, the mechanical properties of the cell are obtained; the computer calculates and processes the changes in the cell surface morphology and mechanical properties, and outputs the imaging diagrams of the cell morphology and mechanical properties at the same time.

[0024] The advantages and beneficial effects of the present invention are as follows:

[0025] Aiming at the deficiencies of the existing cell imaging system that it is impossible to synchronously obtain the morphology and mechanical properties of living cells during imaging, and there are problems such as large volume, complex operation, low resolution, and difficulty in synchronous measurement of multiple parameters, the present invention proposes an optical fiber microbeam probe and system that can realize synchronous imaging of cell morphology and mechanical properties. This cell imaging system has the advantages of multi-parameter synchronous measurement, high resolution, fast imaging speed, small volume, flexible use, and no need for labeling.

[0026] The present invention introduces a metal thin film to enhance the reflectivity of the microbeam, and introduces a nano-tip to improve the resolution of cell morphology and mechanical imaging.

[0027] The present invention proposes an optical fiber microbeam probe system that can realize synchronous imaging of cell morphology and mechanical properties. This system uses the above-mentioned optical fiber microbeam probe as a carrier, utilizes the cavity optomechanical effect of the optical fiber FPI, and integrates all-optical driving and all-optical detection technologies to realize simultaneous high spatio-temporal resolution imaging of the morphology and mechanical properties of living cells. The above-mentioned optical fiber microbeam probe perfectly combines two advantages: namely, the high spatial resolution of the integrated nano-tip and the high time resolution of the optically driven FPI. By using the driving light to excite multiple resonant frequencies, different resonant modes of the microbeam probe are excited. The signals of the first-order resonant mode are used for morphology imaging, and the signals of the second-order resonant mode are used for mechanical property imaging, realizing the simultaneous recording of different characteristic information of cells, and providing a new method for solving the simultaneous high spatio-temporal resolution imaging of the morphology and mechanical properties of living cells.

[0028] The present invention provides a synchronous imaging system for the morphology and mechanical properties of living cells with all-fiber integration. A microbeam probe is integrally fabricated directly on the end face of a single-mode fiber, enabling the driving light and the detection light to directly act on the microbeam probe through the single-mode fiber, replacing the complex and bulky spatial optical path, and achieving both miniaturization and high performance of the cell imaging system. The optical microcavity and the mechanical oscillator are integrally integrated with the fiber without coupling, and the driving and detection efficiencies are high.

[0029] The fiber microbeam probe proposed by the present invention uses a Bragg grating as a high-reflection mirror of the fiber, and the other mirror is realized by coating a metal thin film on the surface of the microbeam probe. In this way, the reflected light intensity of the fiber microbeam probe and the fineness of the reflection spectrum can be greatly improved through energy matching, the quality factor of the optical microcavity can be greatly increased, and the fiber microbeam probe cell imaging system has the advantages of fast imaging speed and high resolution.

[0030] The microbeam proposed by the present invention combines a local hollow pattern and a cuboid structure, greatly improving the resonance frequency of the microbeam probe and the tunability of the resonance mode, and thus enabling the customization of the vibration characteristics of the fiber microbeam probe.

[0031] The present invention has the following advantages compared with CN106501551A: (1) In terms of function realization: The present invention uses the driving light to excite multiple resonance frequencies (dynamic measurement), and different resonance modes of the microbeam probe are excited. The signal of the first-order resonance mode is used for morphology imaging, and the signal of the second-order resonance mode is used for mechanical property imaging, enabling synchronous imaging of the morphology and mechanical properties of living cells. (2) In terms of working mode: The fiber microbeam probe of the present invention integrates all-optical driving and all-optical detection technologies, excites the high-frequency resonance of the microbeam probe, and realizes high-resolution imaging of the morphology and mechanical properties of living cells simultaneously in the dynamic mode. (3) In terms of optical signal detection: The fiber microbeam probe proposed by the present invention uses a Bragg grating as a high-reflection mirror of the fiber, and the other mirror is realized by coating a metal thin film on the surface of the microbeam probe. In this way, the reflected light intensity of the fiber microbeam probe and the fineness of the reflection spectrum can be improved through energy matching, and thus the sensitivity of optical signal detection can be greatly improved. (4) In terms of preparation: The present invention uses femtosecond laser two-photon polymerization technology, focused ion beam (FIB) etching technology, and magnetron sputtering coating technology to prepare the microbeam probe. The manufacturing method is simple and flexible, the materials are diverse (different photoresists can be formulated), the processing accuracy is high, and the preparation of a nano-tip below 1 nm can be realized. Description of the Drawings

[0032] Figure 1 is the preparation flow chart of the fiber microbeam probe sample in the preferred embodiment of the present invention;

[0033] Figure 2 is the structural diagram of the microbeam from different perspectives in the preferred embodiment of the present invention;

[0034] Figure 3 is an optical fiber microbeam probe system that can achieve synchronous imaging of cell morphology and mechanical properties in the present invention;

[0035] Figure 4 is a schematic diagram of the structure and principle of an optical fiber microbeam probe that can achieve synchronous imaging of cell morphology and mechanical properties in the present invention. Specific embodiments

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and detailedly described in conjunction with the accompanying drawings in the embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention.

[0037] As Figure 4 shown, an optical fiber microbeam probe that can achieve synchronous imaging of cell morphology and mechanical properties includes a single-mode optical fiber, a Bragg grating, a support column, a microbeam, and a nano-tip. The single-mode optical fiber includes a core and a cladding outside the core, and has a first end face and a second end face; the Bragg grating is located in the core of the single-mode optical fiber; the first end of the support column is fixed on the second end face of the single-mode optical fiber, one end of the microbeam is fixed to the second end of the support column, the nano-tip is located on the surface of the other end of the microbeam, and the second end face of the single-mode optical fiber, the support column, the microbeam, and the nano-tip together form an optical fiber FPI. A metal thin film is plated on the microbeam and the nano-tip.

[0038] As Figure 1 shown, a method for preparing an optical fiber microbeam probe includes the following steps:

[0039] 1. Take a section of single-mode optical fiber. The single-mode optical fiber includes a core and a cladding outside the core, and has a first end face and a second end face. Use femtosecond laser direct writing technology or phase mask method to prepare a Bragg grating in the core. The femtosecond laser direct writing technology includes, but is not limited to, femtosecond laser point-by-point method, line-by-line method, or surface-by-surface method. During the preparation process, use a spectrometer to monitor the spectrum of the Bragg grating in real time, and the reflectivity of the Bragg grating can be accurately customized. The total length of the Bragg grating is between 200 - 2000 μm.

[0040] 2. Use finite element simulation to design the structure of the microbeam probe. Coat a photoresist solution on the second end face of the single-mode optical fiber, and then use femtosecond laser two-photon polymerization technology to prepare a support column, a microbeam, and a common tip on the cut flat end face of the single-mode optical fiber. A microbeam probe structure composed of the support column, the microbeam, and the common tip is formed in the photoresist solution. Then use photoresist development technology to develop the structure of the microbeam probe to obtain the support column, the microbeam, and the common tip.

[0041] 3. Focus ion beam (FIB) etching or plasma etching is performed on the ordinary tip prepared in the previous step to fabricate the nano-tip, and the diameter of the nano-tip is less than 1 nm.

[0042] 4. A metal thin film is prepared on the surface of the microbeam and the nano-tip by magnetron sputtering coating or evaporation, and the spectrum during the coating process is monitored in real time, so as to realize the matching control between the Bragg grating and the metal thin film. Then, the polished single-mode optical fiber, Bragg grating, support post, microbeam and nano-tip form a complete fiber microbeam probe.

[0043] As Figure 4 shown, a fiber microbeam probe capable of synchronously imaging cell morphology and mechanical properties includes a single-mode optical fiber, a Bragg grating, a support post, a microbeam and a nano-tip. The single-mode optical fiber includes a core and a cladding outside the core, and has a first end face and a second end face; the Bragg grating is located in the core of the single-mode optical fiber; the first end of the support post is fixed on the second end face of the single-mode optical fiber, one end of the microbeam is fixed to the second end of the support post, the nano-tip is located on the surface of the other end of the microbeam, and the second end face of the single-mode optical fiber, the support post, the microbeam and the nano-tip together form an optical fiber FPI. A metal thin film is plated on the microbeam and the nano-tip.

[0044] Figure 2 This is a structural diagram of the microbeam from different perspectives in a preferred embodiment of the present invention, which includes a microbeam substrate, a mass block and a mirror. The microbeam substrate is composed of a locally hollowed-out cuboid and a triangular tip. The locally hollowed-out cuboid enables the mass block to move its position. By moving the position of the mass block or changing the size of the mass block, the overall mass distribution of the microbeam structure can be adjusted, and further, the resonant frequency and resonant mode of the microbeam probe can be changed, realizing the tunability of the resonant frequency and resonant mode of the fiber microbeam probe. The triangular tip matches the conical bottom surface of the nano-tip, making the nano-tip more stable during vibration and reducing the vibration noise generated by lateral torsion. The movement of the position or the change of the size of the mass block can be used for tuning the resonant frequency and resonant mode of the microbeam probe, thereby realizing the customization of vibration characteristics. The mirror is composed of a circular thin sheet and four support rods. The four support rods are used for fixed connection between the circular thin sheet and the microbeam substrate. The circular thin sheet is located directly below the core of the single-mode optical fiber and is used to reflect the light emitted from the core. The light reflected back to the core by the circular thin sheet and the light reflected back to the core by the end face of the single-mode optical fiber interfere with each other, thereby forming the required interference light signal.

[0045] As Figure 3 shown, a method for imaging a fiber microbeam probe system capable of synchronously imaging cell morphology and mechanics includes the following steps:

[0046] 1. The light source emits driving light, which successively passes through an optical isolator, an electro-optic modulator, a coupler, and a circulator and reaches the fiber microbeam probe. Due to photothermal and optomechanical effects, the microbeam probe is excited to vibrate, generating different mechanical resonance modes of the microbeam probe to complete the driving of the microbeam probe.

[0047] 2. The light source emits a detection optical signal, which successively passes through an optical isolator, a coupler, and a circulator and reaches the fiber microbeam probe. The detection optical signal is reflected and transmitted at the second end face of the single-mode fiber. The transmitted light is reflected again on the surface of the microbeam. The two reflected lights form an interference optical signal in the fiber. Since the mechanical resonance of the microbeam probe will cause a periodic change in the cavity length, and thus cause a change in the intensity of the interference optical signal, the monitoring of the mechanical resonance of the microbeam probe can be realized by demodulating and detecting the change in the intensity of the interference optical signal, obtaining state information such as the amplitude-frequency response and phase-frequency response of the microbeam probe in the free vibration state (when far from the cell), and further obtaining the first-order and second-order mechanical resonance modes of the microbeam probe.

[0048] 3. Under the action of a three-dimensional piezoelectric nanoscanner, the nanoscale tip of the microbeam probe vibrating at high speed approaches the cell to be measured and scans its surface. An attractive force will be generated between the nanoscale tip and the cell surface, and this interaction force will affect the resonance state of the microbeam probe, that is, the vibration of the microbeam probe will change with the undulation of the cell surface, and further affect the first-order and second-order resonance states of the microbeam probe.

[0049] 4. The lock-in amplifier detects the changes in amplitude and phase in the first-order resonance mode, and controls the movement of the three-dimensional piezoelectric nanoscanner in the Z-axis direction (up and down) through a feedback controller to keep its first-order amplitude at the initial set value (that is, keep the distance between the nanoscale tip and the cell surface unchanged), record the movement trajectory of the three-dimensional piezoelectric nanoscanner, so as to realize the tracking of the microscopic morphology of the cell surface. At the same time, by detecting the changes in frequency and phase in the second-order resonance mode, the mechanical properties of the cell are obtained. Finally, the computer calculates and processes the changes in the cell surface morphology and mechanical properties (References: [1] Bi Zhuanfang, Shang Guangyi. Nano-mechanical measurement principle and its application of dual-mode atomic force microscopy [J]. Journal of Chinese Electron Microscopy Society, 2019, 38(06): 689-696. [2]. X. Meng; H. Zhang; J. Song; X. Fan; L. Sun; H. Xie. “Broad modulus range nanomechanical mapping by magnetic-drive soft probes,” Nature communications 8(1), 1944, 2017.), and simultaneously outputs the imaging diagrams of the cell morphology and mechanical properties.

[0050] The present invention has the advantages of multi-parameter synchronous measurement, high resolution, fast imaging speed, small volume, flexible use, and no need for labeling.

[0051] S1: Couple a driving optical signal with a first wavelength and a detection optical signal with a second wavelength into the above-mentioned fiber microbeam probe together, so that the driving optical signal drives the microbeam probe to generate resonance.

[0052] S2: Obtain the detection optical signal reflected by the fiber microbeam probe, obtain state information such as the amplitude-frequency response and phase-frequency response of the fiber microbeam probe, and further obtain the first-order and second-order mechanical resonance modes of the microbeam probe.

[0053] S3: The signal of the first-order resonance mode is used for topographic imaging, and the signal of the second-order resonance mode is used for mechanical property imaging.

[0054] Embodiment 1: Fabricate a fiber microbeam probe by using femtosecond laser direct writing technology, femtosecond laser two-photon polymerization technology, focused ion beam (FIB) etching technology, and magnetron sputtering coating technology.

[0055] As Figure 1 shown, the steps are as follows:

[0056] 1. Take an SMF-28 single-mode optical fiber with a core diameter of 8.2 μm and a cladding diameter of 125 μm. After the end face of the optical fiber is cut flat by an optical fiber cutter, fix it on a glass slide.

[0057] 2. Place the glass slide with the fixed SMF-28 single-mode optical fiber on a femtosecond laser microfabrication platform, and use a femtosecond laser with a wavelength of 513 nm and a repetition frequency of 80 MHz to write a Bragg grating in the core of the single-mode optical fiber. The total length of the Bragg grating is 1000 μm.

[0058] 3. Connect a light source and a spectrometer to the single-mode optical fiber on the glass slide through an optical fiber coupler, and monitor its reflection spectrum in real time during the process of writing the Bragg grating. By adjusting the period of the fiber grating, a fiber Bragg grating with a high reflectivity of 90% is prepared.

[0059] 4. Coat a photoresist solution on the second end face of the single-mode optical fiber with the written Bragg grating, and use femtosecond laser two-photon polymerization technology to print out the designed support columns, microbeams, and ordinary tip structures on the fiber end face. The length of the microbeam is 100 μm, the width is 20 μm, and the thickness is 2 μm.

[0060] 5. Develop the printed support columns, microbeams, and ordinary tip structures with a developer to obtain a fiber end face microbeam probe.

[0061] 6. Place the optical fiber end-face microbeam probe prepared in the previous step in a focused ion beam (FIB) etching system, and use focused ion beam (FIB) etching technology to etch the ordinary tip of the microbeam probe so that the diameter of the probe tip reaches 0.8 nm, thereby achieving precise etching preparation of the nanotip.

[0062] 7. After that, the optical fiber microbeam probe is placed in a magnetron sputtering coating instrument, and a gold film is evenly coated on the surface of the optical fiber microbeam probe. The thickness of the gold film is controlled at 20nm. At this point, the flattened single-mode optical fiber, the Bragg grating in the fiber core, the support column, the microbeam and the nano-needle tip constitute a complete optical fiber microbeam probe. Figure 4 Schematic diagram of the structure and principle of the fiber optic microbeam probe that can achieve simultaneous imaging of cell morphology and mechanical properties.

[0063] It should also be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, commodity or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, commodity or device. In the absence of more restrictions, the elements defined by the sentence "comprises a ..." do not exclude the existence of other identical elements in the process, method, commodity or device including the elements.

[0064] The above embodiments should be understood to be only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the contents of the present invention, technicians can make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

Claims

1. An optical fiber microbeam probe capable of realizing simultaneous imaging of cell morphology and mechanical properties, characterized in that: The invention comprises a single-mode optical fiber, a Bragg grating, a support column, a micro-beam and a nano-needle tip. The single-mode optical fiber comprises a core and a cladding outside the core, and has a first end face and a second end face. The Bragg grating is located in the core of the single-mode optical fiber. The first end of the support column is fixed to the second end face of the single-mode optical fiber, one end of the micro-beam is fixed to the second end of the support column, and the nano-needle tip is located on the surface of the other end of the micro-beam. The second end face of the single-mode optical fiber, the support column, the micro-beam and the nano-needle tip together form an optical fiber Fabry-Perot interferometer FPI, and an optical microcavity is formed between the second end face of the single-mode optical fiber and the micro-beam.

2. The optical fiber microbeam probe capable of realizing synchronous imaging of cell morphology and mechanical properties according to claim 1, characterized in that: The micro beam and the nano needle tip are coated with a metal film.

3. The optical fiber microbeam probe capable of realizing synchronous imaging of cell morphology and mechanical properties according to claim 2, characterized in that: The metal film is a gold, silver or copper metal film.

4. An optical fiber microbeam probe capable of realizing simultaneous imaging of cell morphology and mechanical properties according to any one of claims 1 to 3, characterized in that: The microbeam includes a microbeam matrix, a mass block and a reflector. The microbeam matrix is ​​a partially hollowed-out thin sheet structure, at least one end of which is a triangular tip, which is used to fix the nano needle tip. The mass block is arranged at the hollowed-out part of the microbeam matrix so that the mass block can move horizontally along the microbeam matrix. The reflector is arranged on the surface of the microbeam matrix.

5. The method for preparing the optical fiber microbeam probe capable of realizing simultaneous imaging of cell morphology and mechanical properties according to any one of claims 1 to 4, comprising the following steps: First, a Bragg grating is prepared in the core of a single-mode optical fiber. Second, support columns, microbeams and ordinary needle tips are prepared on the flattened end face of the single-mode optical fiber using femtosecond laser two-photon polymerization technology. Third, the ordinary needle tip from the previous step is etched to achieve the preparation of a nano-needle tip. Finally, a metal film is prepared on the surface of the microbeam and nano-needle tip.

6. The method for preparing the optical fiber microbeam probe capable of realizing synchronous imaging of cell morphology and mechanical properties according to claim 5, characterized in that: The method for preparing the Bragg grating is to prepare the Bragg grating in the fiber core using a femtosecond laser direct writing technique or a phase mask method.

7. The method for preparing the optical fiber microbeam probe capable of realizing synchronous imaging of cell morphology and mechanical properties according to claim 5, characterized in that: The method for preparing the support column, microbeam and common needle tip comprises the following steps: firstly coating a polymer photoresist solution on the end face of an optical fiber, then optimizing the structural dimensions and shapes of the support column, microbeam and common needle tip by using finite element simulation, then printing the designed support column, microbeam and common needle tip structures on the second end face of the optical fiber by using femtosecond laser two-photon polymerization technology, and finally developing the printed structures by using photoresist development technology.

8. The method for preparing the optical fiber microbeam probe capable of realizing synchronous imaging of cell morphology and mechanical properties according to claim 5, characterized in that: The metal film is prepared on the surface of the micro-beam and the nano-needle tip by magnetron sputtering coating or evaporation.

9. A system for realizing simultaneous imaging of cell morphology and mechanical properties constructed by using the optical fiber microbeam probe according to any one of claims 1 to 4, characterized in that: It includes optical fiber micro-beam probe, driving light source, detection light source, signal generator, electro-optic modulator, three-dimensional piezoelectric nano-scanner, feedback controller, computer, circulator, coupler, tunable optical filter, photodetector, and analyzer; The driving light source emits driving light, and the signal generated by the signal generator modulates the driving light through the electro-optical modulator. The modulated driving light reaches the optical fiber micro-beam probe through the coupler and the circulator, and excites the micro-beam probe to generate resonance. The detection light source emits a detection light signal, which passes through the coupler and the circulator in sequence and reaches the optical fiber microbeam probe; A three-dimensional piezoelectric nanoscanner is arranged below the optical fiber microbeam probe, and living cells or tissues are placed on the three-dimensional piezoelectric nanoscanner. When the nano needle tip approaches the living cell or tissue and scans on its surface, the mutual attraction between the needle tip and the cell or tissue surface will affect the resonance state of the microbeam probe. The detection light signal is reflected and transmitted at the second end face of the single-mode optical fiber, and then reflected again at the surface of the micro-beam. The two reflected lights form an interference light signal in the optical fiber, and then enter the analyzer after passing through the circulator, tunable optical filter and photodetector, thereby obtaining the first-order and second-order resonance states of the micro-beam probe. The analyzer is connected to a computer, and the feedback controller is connected to the computer and the three-dimensional piezoelectric nanoscanner respectively; The analyzer detects the changes in amplitude and phase in the first-order resonant state, and controls the movement of the three-dimensional piezoelectric nanoscanner in the Z-axis direction through a feedback controller to keep its first-order amplitude at the initial set value, and records the movement trajectory of the three-dimensional piezoelectric nanoscanner, thereby tracking the microscopic morphology of the cell surface; the mechanical properties of the cells are obtained by detecting the changes in frequency and phase in the second-order resonant state; the computer calculates and processes the changes in the cell surface morphology and mechanical properties, and simultaneously outputs imaging images of the cell morphology and mechanical properties.

10. The system for realizing simultaneous imaging of cell morphology and mechanical properties according to claim 9, characterized in that: The analyzer is an electrical spectrum analyzer, a vector network analyzer or a phase-locked amplifier.

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