A terahertz near-field microscope that can be used in combination with fluorescence and its usage method

Abstract

The present invention discloses a terahertz near-field microscope that can be used in conjunction with fluorescence and its usage method, which relates to the field of near-field microscopes and includes: a terahertz unit, an atomic force microscope unit, a fluorescence microscope unit, and a processing unit. The terahertz unit includes: a femtosecond laser light source, an optical fiber beam splitter, an optical fiber delay line, a transmitting antenna, a first focusing parabolic mirror, a second focusing parabolic mirror, and a receiving antenna; the atomic force microscope unit includes: an atomic force probe, a quadrant laser emitter, a quadrant detector, and a sample stage; the fluorescence microscope unit includes: a high-pressure mercury lamp, an excitation filter, a first dichroic mirror, a fluorescence objective lens, an illumination light source, a collimating lens, a second dichroic mirror, an emission filter, and a fluorescence camera; the present invention utilizes the advantages of high penetration and super-resolution imaging of the terahertz near-field microscope to specifically solve the problems in measuring biological samples with a fluorescence microscope.

Description

Technical Field

[0001] The present invention relates to the field of near-field microscopes, and in particular, to a terahertz near-field microscope that can be used in combination with fluorescence and a method for using the same. Background Art

[0002] A fluorescence microscope is an optical microscope widely used in the field of biomedical imaging. With the invention of dyes, fluorescence microscopy has greatly promoted the development of immunology and cell biology. Compared with traditional microscopy, fluorescence microscopy has high specificity and sensitivity, and can detect substances with extremely low content with extremely low concentrations of fluorescent dyes. Moreover, with the continuous invention of fluorescent dyes, the range of substances that can be detected is further expanding.

[0003] Most commonly used fluorescence microscopes at present are inverted fluorescence microscopes, and their composition usually includes an illumination light source, which becomes violet light after passing through a filter, also known as an excitation light source; a dichroic mirror, which is used to reflect the illumination light source and transmit the fluorescence excited by the sample at the same time; a high-power objective lens, which is used to focus the excitation light source and collect fluorescence at the same time; and an array detector (such as a CCD, etc.), which is used to observe and collect fluorescence.

[0004] However, although the existing fluorescence microscopy technology can achieve the observation of samples such as biological cells, it is limited by the optical diffraction limit and is difficult to achieve higher-resolution observations, and it is difficult to achieve the observation of the fine structure on the cell surface and the fine structure inside the cell. Summary of the Invention

[0005] The object of the present invention is to overcome the bottleneck problems such as the optical resolution limit of the current fluorescence microscope, the inability to obtain parameters such as the cell surface structure and mechanics when irradiating biological samples, and the inability to image the internal structure of cells.

[0006] To achieve the above object of the invention, the present invention provides a terahertz near-field microscope that can be used in combination with fluorescence. The terahertz near-field microscope includes: a terahertz unit, an atomic force microscope unit, a fluorescence microscope unit, and a processing unit, wherein:

[0007] The terahertz unit includes: a femtosecond laser light source, an optical fiber beam splitter, an optical fiber delay line, a transmitting antenna, a first focusing parabolic mirror, a second focusing parabolic mirror, and a receiving antenna; the femtosecond laser light source is used to generate a laser, which is divided into a first transmitted light and a first received light by the optical fiber beam splitter. The first transmitted light is delayed by the optical fiber delay line and then focused and incident on the transmitting antenna to generate a terahertz wave. The optical fiber delay line can also be used to adjust the distance between the transmitting antenna and the receiving antenna to match the optical path of the fluorescence objective lens body; the terahertz wave is focused by the first focusing parabolic mirror to the region between the atomic force probe tip and the sample, generating a scattered terahertz near-field signal. The terahertz near-field signal is focused by the second focusing parabolic mirror, then collimated and emitted and focused and incident on the receiving antenna. The terahertz near-field signal and the first received light act on the receiving antenna to make the carriers move directionally and then generate a first electrical signal. The first electrical signal is transmitted to the processing unit and after being processed by phase-locked demodulation, the terahertz near-field spectral image of the sample is obtained;

[0008] The atomic force microscope unit includes: an atomic force probe, a quadrant laser emitter, a quadrant detector, and a sample stage; the quadrant laser emitter is used to emit a laser to the tip cantilever of the atomic force probe, and the laser is reflected back to the quadrant detector by the tip cantilever. The sample stage is a hollow stage, and the sample is suspended in the hollowed-out area of the sample stage;

[0009] The fluorescence microscope unit includes: a high-pressure mercury lamp, an excitation filter, a first dichroic mirror, a fluorescence objective lens, an illumination light source, a collimating lens, a second dichroic mirror, an emission filter, and a fluorescence camera;

[0010] The illumination light source is used to emit illumination light. The illumination light is collimated by the collimating lens and then enters the second dichroic mirror. The illumination light is reflected by the second dichroic mirror and then enters the fluorescence objective lens. The illumination light is focused by the fluorescence objective lens and then irradiates the sample on the sample stage to illuminate the sample surface; the high-pressure mercury lamp is used to generate radiation light in the ultraviolet to infrared band. The radiation light is filtered by the excitation filter to generate a preset wavelength characteristic light source. The preset wavelength characteristic light source is reflected by the first dichroic mirror and then enters the fluorescence objective lens and is focused on the sample on the sample stage to excite fluorescence. The fluorescence is reflected and then sequentially passes through the fluorescence objective lens, the first dichroic mirror, the second dichroic mirror, and the emission filter and then enters the fluorescence camera. The fluorescence camera transmits the collected fluorescence data information to the processing unit, and the processing unit processes the information collected by the fluorescence camera and obtains the fluorescence image of the sample.

[0011] Among them, the emission filter is used to filter the short-wavelength light emitted by the mercury lamp and only transmit the fluorescence generated by the sample. The generated fluorescence wavelength is longer than the wavelength of the mercury lamp passing through the excitation filter. The emission filter is a short-wave cut-off and long-wave pass filter.

[0012] Among them, the present invention proposes a technical solution that can be used in combination with a terahertz near-field microscope, which can simultaneously achieve many advantages such as the high penetration of terahertz, the high resolution of the atomic force microscope, and the cell labeling observation ability of the fluorescence microscope; at the same time, the microscope in the present invention can also detect and measure the interaction force between active molecules on the cell membrane surface; therefore, the cell structure information that can be observed by the microscope in the present invention will be richer.

[0013] The terahertz near-field microscope is a new type of super-resolution detection instrument that combines terahertz time-domain spectroscopy technology and atomic force microscope, and has both the high penetration of terahertz waves and the super-resolution ability of the atomic force microscope. The vibration and rotation energy levels of many biological macromolecules are also in the terahertz band, which makes the terahertz near-field microscope have unique advantages in the biological imaging of living cells. It can not only achieve super-resolution observation of the cell surface structure, but also observe the internal structure of cells at a certain distance from the cell surface.

[0014] The terahertz near-field microscope is a new type of scattering near-field optical technology that combines terahertz time-domain spectroscopy technology and near-field optical microscope. However, in the traditional terahertz near-field microscope, the positions of the terahertz emission and reception antennas are fixed, which results in the inability to flexibly adjust the positions of the emission and reception antennas in the overall optical path according to the sample structure, size or other devices used in combination. The terahertz near-field microscope in this system has been improved in the delay part, and the distance between the emission and reception antennas can be adjusted according to the overall structure of the fluorescence microscope.

[0015] The specific structure of this delay line is a double-layer structure, and the optical path is encapsulated and connected by means of optical fiber series connection. The upper part is a short-stroke fast-moving linear motor with a maximum moving speed of 1 m / s and a maximum stroke of 12 cm (corresponding optical path is 400 ps). The lower part is a slow-step long-stroke stepper motor with a stepping motion, a moving speed < 5 cm / s, and a maximum stroke of 60 cm. Both are optical fiber-encapsulated delay lines. The short-stroke linear motor is fixed on the long-stroke stepper motor, and the two are connected together through an adapter plate. The scanning speed of the short-stroke linear motor is 10 waveforms per second, and the sampling length of each waveform is 50 ps. It integrates a grating ruler with an accuracy of 100 nm for stepping motion in the near-field test environment. The adjustable range of the long-stroke stepper motor is 0 - 2000 ps, which is used to adjust the distance between the emission and reception antennas. When specifically used, the two do not move simultaneously. Specifically, when performing fast scanning, the upper linear motor is used, and when performing near-field imaging, the upper linear motor is also used. Only when it is necessary to adjust the distance between the emission and reception antennas, the upper linear motor does not move, and only the long-stroke stepper motor is used to adjust the optical path between the emission and reception antennas. It is expressed by the formula as follows:

[0016] Assume that the distance to be adjusted between transmission and reception is Δx, and the optical path inside the long-travel delay line is 4 times the round-trip optical path. Therefore, the distance that needs to be adjusted for the long-travel delay line is Δx / 4.

[0017] The traditional near-field optical system microscope is developed on the basis of the atomic force microscope. Generally, the support feet of the scanning head part of this microscope are not high enough to install a parabolic mirror for focusing. Although the commercially available terahertz near-field microscope can install a focusing parabolic mirror, the size of the focusing mirror is very small, making it difficult to effectively collect more incident and scattered terahertz wave signals. In the atomic force microscope scanning head part of the present invention, after the transformation of the support feet and the probe height, it has been able to install a focusing parabolic mirror with a height of 25 mm, etc., and can effectively collect incident and scattered terahertz wave signals.

[0018] For an ordinary inverted fluorescence microscope, the accuracy of its scanning stage often stays in the micron-level accuracy range. However, due to the combined use requirement with the near-field microscope, its accuracy needs to meet the needs of the scanning head of the atomic force microscope and must reach the nanometer level. At the same time, it can transmit the fluorescence signal, and the size of the hollow part is sufficient to meet the free switching between fluorescence objective lens groups. Therefore, in the present invention, a hollow nanometer-level high-precision three-dimensional piezostage is specifically designed to meet the above requirements.

[0019] A method of combining a terahertz near-field microscope and an inverted fluorescence microscope provided by the present invention utilizes the advantages of the terahertz near-field microscope such as high penetrability and super-resolution imaging, and specifically solves the problems when measuring biological samples with a fluorescence microscope, providing a new way for the high-resolution multi-parameter synchronous detection of biological samples.

[0020] Preferably, the atomic force microscope unit further includes at least one support and a housing. The lower end of the support is connected to the sample stage, and the upper end of the support is connected to the lower end of the housing. The quadrant laser emitter and the quadrant detector are located inside the housing. Holes are respectively opened on the housing for adjusting the laser emitted by the quadrant laser emitter and for adjusting the laser received by the quadrant detector. An emission adjustment knob and a reception adjustment knob are installed at the holes to ensure that the quadrant laser accurately irradiates the sample surface and is accurately received at the center of the detector.

[0021] Among them, the housing is used to protect the internal circuits and devices, and the support is used to support the components above the sample stage to form a certain space, which is used for the atomic force probe to measure the sample.

[0022] Preferably, the atomic force microscope unit further includes a first observation camera, which is used to obtain the real-time position of the atomic force probe. A hole for the first observation camera to observe is opened above the housing.

[0023] Among them, the traditional atomic force microscope does not have a terahertz part and a fluorescence display part, and there are many angles and spaces for observation, and there is no dedicated observation hole for observation. Since the microscope in the present invention adds a terahertz part and a fluorescence microscope part in addition to the traditional atomic force microscope part, an observation hole is opened above the outer shell to ensure that the observation camera directly above can observe the distance between the tip and the sample through the observation hole. Only when the tip touches the sample can relevant tests be carried out, and at the same time, the sample boundary can be observed in real time and the scanning area can be specified.

[0024] Preferably, the height of the support is greater than the height of the first focusing parabolic mirror and the height of the second focusing parabolic mirror respectively. Since the support feet of the traditional atomic force microscope are too close to the sample surface, an additional optical path cannot be added, and the combination with terahertz cannot be realized. Therefore, the present invention improves the traditional atomic force microscope, increases the height of the support of the atomic force microscope, and in order to achieve the focusing of the focusing parabolic mirror, the height of the support needs to be greater than the height of the first focusing parabolic mirror and the height of the second focusing parabolic mirror respectively.

[0025] Preferably, the terahertz near-field microscope further includes a current amplifier and a lock-in amplifier. The first electrical signal is input into the current amplifier for amplification processing. The amplification factor of the current amplifier is 20MV / A. The amplified signal is input into the lock-in amplifier for demodulation, and the signal after the lock-in amplifier demodulation processing is input into the processing unit.

[0026] Among them, since the electrical signal obtained by scattering is a very weak electrical signal, it cannot be directly and effectively observed with current electrical measuring instruments. Therefore, the present invention uses a current amplifier to amplify the weak electrical signal for convenient observation. After the weak electrical signal passes through the current amplifier, while the effective signal is amplified, the surrounding noise signals will also be amplified. Therefore, the lock-in function is used to suppress the noise signals, so as to achieve the purpose of only amplifying the effective signal, and thus the effective amplification and observation of the weak electrical signal are realized as a whole.

[0027] Preferably, the demodulation frequencies of the 4 signal channels in the lock-in amplifier are set. The 4 signal channels are respectively connected to the corresponding 4 imaging channels. The 4 imaging channels respectively output atomic force topography imaging, first-order near-field imaging, second-order near-field imaging and third-order near-field imaging. Among them, the demodulation order n can also be independently set for each channel. At this time, the imaging corresponds to the nth-order near-field imaging, where n is an integer greater than 1.

[0028] Among them, there are 4 channels for phase locking. One of them is the channel for the atomic force topography itself, which is used to compare the image scanned by the atomic force itself with the images of the near-field signals of each order later, so as to more effectively illustrate the superiority of the near-field signals and be able to see the parts that cannot be seen by the atomic force microscope. The other three of the 4 channels for phase locking are near-field demodulation channels. These three channels can actually demodulate different near-field orders. The higher the order, the weaker the signal. Therefore, generally, the near-field signals of orders 1, 2, and 3 are directly demodulated because the signals after the 3rd order are very weak and the observed images are not clear either.

[0029] Regarding the tip demodulation principle of the lock-in amplifier, it is necessary to explain it in detail here. Assuming that the sample and the tip are regarded as a dipole model, we can obtain the following formula:

[0030] d = z + r = A[1 + cos(2πΩt)] (1)

[0031] Among them, d is the distance between the center of the tip curvature radius and the sample surface, r is the tip curvature radius, z is the distance between the bottom of the tip and the sample, A represents the amplitude of the probe, Ω is the tip demodulation frequency, and t is the time parameter. From equation (1), it can be seen that the probe vibration makes the dipole moment become a function of time, which provides the basis for the demodulation of the scattered wave. At this time, assuming that α is the tip polarizability of the probe and β is the reflection coefficient related to the sample, the following formulas can be obtained respectively:

[0032] α = 4πr 3 (ε t - 1) / (ε t + 2) (2)

[0033] β = (ε s - 1)(ε s + 1) (3)

[0034] Among them, ε t is the dielectric constant of the tip of the probe, ε s is the dielectric constant of the sample. Considering that the incident laser is linearly polarized light and is P light at this time, the dipole polarizability between the tip and the sample is expressed as α eff-tip . Assuming that E s represents the scattered light electric field intensity and E i represents the incident light electric field intensity, the following formulas are respectively available:

[0035]

[0036] E s ∝ α eff-tip (1 + γ) 2 E i (5)

[0037] where γ is the reflection coefficient of the sample surface. The above formula relates the relationship between the scattered light intensity and the incident light intensity. According to the analysis, the scattered light intensity is related to the dielectric constant between the tip and the sample. Among them, α eff-tip satisfies the Dirichlet condition, so it can be expressed in exponential form:

[0038]

[0039] At the same time, F n represents the Fourier coefficients of each harmonic component, F 0 is the Fourier coefficient of the 0th harmonic component, F 1 is the Fourier coefficient of the first harmonic component, F 2 is the Fourier coefficient of the second harmonic component. Therefore, j is the imaginary unit. For a given sample and probe ε s and ε t are both determined quantities, then there is:

[0040]

[0041] Among them, σ is a fixed value corresponding to (1 + γ) 2 The is the phase angle of the nth harmonic component. From equation (7), it can be seen that E s can be decomposed into a series of harmonic components relative to Ω, which is also the principle of the lock-in amplifier to demodulate each harmonic component. At this time, the near-field signal obtained by demodulating the fundamental frequency component contains noise components, and the higher-order demodulation contains less noise components.

[0042] Preferably, the terahertz unit is located above the sample stage, and the fluorescence microscope unit is located below the sample stage. The above structure is specially designed. Among them, the terahertz unit is above because the atomic force scanning head part needs to rely on its own gravity and rely on three support feet to achieve stable placement. If this part is inverted, it cannot be fixed by gravity; secondly, the probe of the atomic force microscope realizes the high-frequency tapping between the tip and the sample when scanning the sample. If it is inverted, this tapping measurement will bring great errors, thus unable to achieve the precise measurement of the atomic force itself, let alone the accuracy of the subsequent near-field demodulation signal; finally, the measurement of the atomic force probe is realized by contacting the sample surface, while the fluorescence measurement is through the transparent substrate focusing measurement. If it is inverted, the sample is placed on the substrate, and the atomic force probe cannot contact the sample surface, so it cannot be accurately measured.

[0043] Preferably, the sample stage is provided with a movement control unit for controlling the sample stage to move in three directions of the X-axis, Y-axis and Z-axis respectively. Using the movement control unit is convenient for adjusting the position of the sample, and it is also convenient for placing, taking out and replacing the sample.

[0044] To achieve the above-mentioned invention object, the present invention also provides a method for using a terahertz near-field microscope that can be used in combination with fluorescence, and the method includes:

[0045] Testing steps:

[0046] Step 1: Suspend the test sample in the hollow area of the sample stage; the sample is placed on a substrate, which is a fused silica with a thickness of 500 um. An indium tin oxide thin film with a thickness of 1 um is plated on the upper surface of the fused silica, and its core components are 90% In 2 O 3 and 10% SnO 2 . Due to the effect of the coating, this substrate can effectively reflect the near-field terahertz signal, and the effective reflectivity is as high as more than 80%. At the same time, this substrate has high transmittance in the visible light to infrared range, so it can be used for transmitting fluorescence signals at the same time.

[0047] Step 2: Turn on the femtosecond laser light source;

[0048] Step 3: Turn on the quadrant laser emitter, adjust the quadrant laser emitter so that the laser emitted by the quadrant laser emitter is reflected back to the exact center of the quadrant detector by the tip cantilever, and then the atomic force probe advances until it touches the surface of the test sample;

[0049] Step 4: Adjust the transmitting antenna so that the terahertz wave is focused on the tip of the atomic force probe through the first focusing parabolic mirror. The receiving antenna outputs the first electrical signal to the current amplifier, and after being amplified by the current amplifier, it is input to the input end of the lock-in amplifier;

[0050] Step 5: Turn on the lock-in amplifier, set the demodulation frequencies of the 4 signal channels in the lock-in amplifier. The 4 signal channels are respectively connected to the corresponding 4 imaging channels, and the 4 imaging channels respectively output atomic force topography imaging, first-order near-field imaging, second-order near-field imaging diagram and third-order near-field imaging. Each channel can also independently set the demodulation order n, and at this time the imaging corresponds to the nth-order near-field imaging, where n is an integer greater than 1;;

[0051] Step 6: Turn on the high-pressure mercury lamp, illumination light source and fluorescence camera;

[0052] Step 7: Use the fluorescence objective lens to find the position of the test sample corresponding to the tip of the atomic force probe, and then take the fluorescence image of this position;

[0053] Step 8: After taking the fluorescence image, switch the objective lens group in the fluorescence objective lens from high magnification to low magnification, and thus complete the testing steps;

[0054] Measurement steps:

[0055] After the test steps are completed, replace the test sample with the device under test for measurement.

[0056] Among them, after taking the fluorescence image in step 8, the objective lens group in the fluorescence objective lens needs to be switched from high magnification to low magnification, and then the measurement step is carried out. The purpose of doing this is to start scanning the terahertz near-field image of the area at this point after switching the objective lens group in the fluorescence objective lens from high magnification to low magnification. Because the terahertz near-field scanning is carried out in a very small area of the sample, and the sample stage will perform three-dimensional movement. To ensure the safety of the atomic force tip and the highest magnification objective lens, the objective lens here must be switched to the lowest magnification to ensure a certain spatial distance from the sample.

[0057] Preferably, step 7 includes:

[0058] Step 7.1: Select the low magnification objective lens group in the objective lens group of the fluorescence objective lens to preliminarily observe and obtain the sample position range corresponding to the atomic force probe tip;

[0059] Step 7.2: Based on this range, switch the low magnification objective lens group in the fluorescence objective lens to the high magnification objective lens group, find the sample position corresponding to the atomic force probe tip, and then take the fluorescence image of this position.

[0060] Among them, first look with the low magnification objective lens to find a suitable area, then switch to the high magnification objective lens to look, and then accurately find the sample position corresponding to the atomic force probe tip, which can improve the search efficiency.

[0061] Preferably, the method further includes:

[0062] Replacement steps:

[0063] Step a: Lower the fluorescence objective lens by a first distance value. The purpose of this step is to ensure a sufficient safety distance from the sample;

[0064] Step b: Lower the sample stage by a second distance value, where the first distance value is greater than the second distance value; the purpose of this step is to move the sample surface away from the atomic force probe. The step order of step a and step b cannot be changed, and the lowering distance of the objective lens must be greater than the lowering distance of the three-dimensional piezoelectric scanning stage, otherwise the probe or the objective lens will be damaged;

[0065] Step c: Take out the original sample in the sample stage and put a new sample into the sample stage.

[0066] One or more technical solutions provided by the present invention have at least the following technical effects or advantages:

[0067] The present invention can address the bottleneck problems of current fluorescence microscopes, such as the optical resolution limit and the inability to obtain parameters such as the surface structure and mechanics of biological samples when irradiating biological samples, and the inability to image the internal structure of cells. By leveraging the advantages of terahertz near-field microscopes, such as high penetrability and super-resolution imaging, the present invention specifically solves the problems encountered when measuring biological samples with fluorescence microscopes, providing a new approach for the simultaneous high-resolution multi-parameter detection of biological samples.

[0068] The present invention combines terahertz waves with a fluorescence microscope under a sample stage by introducing terahertz waves into a near-field microscopy system, achieving in-situ real-time imaging of cells or biological tissues. Parameters such as the surface topography and mechanical properties of cells are obtained through terahertz near-field spectroscopy, and fluorescence information of cells or tissues is obtained through a fluorescence microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of the present invention, and do not limit the embodiments of the present invention;

[0070] Figure 1 It is a schematic diagram of the composition of a terahertz near-field microscope that can be used in conjunction with fluorescence;

[0071] Figure 2 It is a schematic diagram of the working principle of the microscope in the present invention;

[0072] Figure 3 It is a schematic diagram of the sample replacement method of the display mirror in the present invention;

[0073] Among them, 1 - femtosecond laser light source, 2 - fiber optic beam splitter, 3 - fiber optic delay line, 4 - transmitting antenna, 5a - first focusing parabolic mirror, 5b - second focusing parabolic mirror, 6 - atomic force probe, 7 - quadrant laser emitter, 8 - quadrant detector, 9 - receiving antenna, 10 - sample stage, 11 - high-pressure mercury lamp, 12 - excitation filter, 13 - fluorescence objective lens, 14 - illumination light source, 15 - first dichroic mirror, 16 - second dichroic mirror, 17 - emission filter, 18 - fluorescence camera, 19 - processor, 20 - collimating lens. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0074] In order to more clearly understand the above objects, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the drawings and specific embodiments. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0075] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described within the scope hereof. Therefore, the scope of protection of the present invention is not limited by the specific embodiments disclosed below.

[0076] Embodiment 1

[0077] Please refer to Figure 1 , Figure 1 , which is a schematic diagram of the composition of a terahertz near-field microscope that can be used in combination with fluorescence. A terahertz near-field microscope that can be used in combination with fluorescence in this embodiment includes: a 1550 nm femtosecond laser light source 1, an optical fiber beam splitter 2, an optical fiber delay line 3, a transmitting antenna 4, a first focusing parabolic mirror 5a, a second focusing parabolic mirror 5b, an atomic force probe 6, a quadrant laser emitter 7, a quadrant detector 8, a receiving antenna 9, a sample stage 10, a high-pressure mercury lamp 11, an excitation filter 12, a first dichroic mirror 15, a fluorescence objective lens 13, an illumination light source 14, a collimating lens 20, a second dichroic mirror 16, an emission filter 17, a fluorescence camera 18, and a processor 19. The laser emitted by the 1550 nm femtosecond laser light source 1 is divided into two paths of light for emission and reception by the optical fiber beam splitter 2. The emitted light is incident on the transmitting antenna 4 after passing through the optical fiber delay line 3 to generate terahertz waves. The terahertz waves are focused on the atomic force microscope probe 6 by the first focusing parabolic mirror 5a. The laser emitted by the quadrant laser emitter 7 enters the quadrant receiver 8 after being reflected by the probe cantilever. The terahertz near-field scattering signal enters the receiving antenna 9 after being scattered by the tip. An electrical signal is generated based on the terahertz near-field scattering signal and the received laser by the receiving antenna, and the electrical signal is transmitted to the processor for processing.

[0078] The test sample is placed on the hollow sample stage 10. The high-pressure mercury lamp 11 emits high-intensity characteristic spectral lines. The characteristic spectral lines pass through the excitation filter 12 and are reflected by the first dichroic mirror 15 and then enter the fluorescence objective lens 13 to irradiate the sample to excite fluorescence. The fluorescence enters the fluorescence camera 18 after being reflected through the second dichroic mirror 16 and the emission filter 17. The fluorescence camera transmits the collected fluorescence image information to the processor 19 for processing. Among them,

[0079] Among them, the excitation filter 12: The light emitted by the mercury lamp contains spectral lines in all bands from ultraviolet to infrared. It is a broadband light source. The excitation filter will filter out signals in other bands and only pass short-band excitation light such as ultraviolet to irradiate the sample.

[0080] The first dichroic mirror 15: Emits short-wavelength excitation light and transmits the fluorescence reflected back by the sample. Generally speaking, the wavelength of fluorescence is greater than that of the excitation light.

[0081] Illumination light source 14: This light source is used to illuminate the sample itself to facilitate the observation of the sample area. This light source is a divergent light source, so a collimating lens is required for optical path collimation.

[0082] Collimating lens 20: Collimates the light source of the divergent light source 14 and then irradiates it onto the sample.

[0083] Second dichroic mirror 16: This mirror can reflect the illumination light source of 14 to illuminate the sample, and at the same time can transmit the fluorescence signal.

[0084] Emission filter 17: The emission filter can effectively filter out stray light signals in other wavelength bands and only transmit the effective fluorescence part of the signal.

[0085] In the present invention, by introducing terahertz waves into the near-field microscopy system and combining it with the fluorescence microscope under the sample stage, in-situ real-time imaging of cells or biological tissues is achieved. Terahertz near-field spectroscopy obtains parameters such as the surface topography and mechanical properties of cells. The fluorescence microscope obtains the fluorescence information of cells or tissues. Among them, the three-dimensional topography measurement by the atomic force probe is realized through the real-time distance feedback between the tip and the sample. The laser emitted by the quadrant laser emitter 7 is reflected back into the quadrant detector 8 after irradiating the tip cantilever. In this way, the tip jitter will cause the laser reflected into the quadrant detector 8 to hit different positions on the quadrant detector 8. By recording this different position, the distance between the tip and the sample can be recorded in real time, thus realizing three-dimensional measurement.

[0086] A hollow area or through hole is provided in the middle of the sample stage 10. The purpose is to facilitate suspending the sample. The goal of suspension is to facilitate the observation by the atomic force microscope and terahertz above, and at the same time facilitate the observation by the fluorescence microscope part below. The suspension method can use a clamping member for clamping or other similar fixing components, and the embodiments of the present invention do not make specific limitations.

[0087] The emitted light generates terahertz after passing through the emission antenna 4, and then reaches the receiving antenna 9 after being scattered by the tip. When the received light irradiates the receiving antenna 9, free carriers are generated inside the receiving antenna, and at this time the movement directions are chaotic. At this time, the scattered terahertz reaches the receiving antenna 9, and the electric field of the terahertz will cause the carriers inside the antenna 9 to move in a directional manner, thus generating a regular electrical signal, so as to achieve the purpose of detecting the scattered terahertz wave. The scattered terahertz wave itself is generated by the interaction between the sample and the tip, so the purpose of detecting the sample is achieved. The purpose of the detection light is to generate free carriers inside the detection antenna, and the scattered terahertz is to make these free carriers move in a directional manner to facilitate the readout of the electrical signal, and the read electrical signal is used as the imaging signal.

[0088] Among them, in the traditional atomic force microscope, since the supporting feet are too close to the sample surface, it is impossible to externally add an additional optical path. The microscope in this embodiment is a terahertz near-field - fluorescence combined atomic force microscope. It is necessary to transform the atomic force itself. First, in order to introduce and focus terahertz waves, the supporting parts of the atomic force microscope, such as the supporting feet, need to have a certain height. The specific height is determined by the size of the parabolic mirror. The height of the supporting feet needs to be greater than the height of the parabolic mirror so that the parabolic mirror can be placed in the space between the atomic force probe and the sample stage.

[0089] The sample stage in this embodiment is a three-dimensional sample stage, that is, the sample stage can move in the X / Y / Z axis directions, which is convenient for adjusting the position of the sample.

[0090] The number of supporting feet in this embodiment can be one or more, preferably 3. The supporting feet can be electric supporting feet, which are convenient for adjusting the height. And the 3 supporting feet can achieve adjustments in three direction dimensions, thereby realizing the three-dimensional movement of the probe. At the same time, the triangular support has stability, which is very important for the detection of atomic force microscopes at the micro-nano scale.

[0091] At the same time, the present invention is an improvement on the basis of the traditional atomic force microscope. It is improved by increasing the height of the supporting feet and opening corresponding observation holes. However, simply raising the supporting feet of the atomic force microscope will cause a change in the height of the atomic force probe, thus unable to achieve the purpose of fluorescence combination. Therefore, the height raised by the supporting feet must be equal to the descending height of the atomic force tip, which is equivalent to the height raised by the supporting feet compared with the original being equal to the descending height of the atomic force tip, and the height of the atomic force tip remains unchanged compared with the initial state.

[0092] An observation hole is opened directly above the shell of the traditional atomic force microscope. Through the observation camera at the observation hole, the distance between the tip and the sample can be observed. Only when the tip touches the sample can relevant tests be carried out. At the same time, the sample boundary can be observed in real time and the scanning area can be specified.

[0093] Embodiment 2

[0094] On the basis of Embodiment 1, please refer to Figure 2 , Figure 2 , which is a schematic diagram of the working principle of the microscope in the present invention. Embodiment 2 of the present invention provides the usage method of the above microscope, specifically as follows:

[0095] First, the femtosecond laser is split and then connected to the transmitting (TX) and receiving antennas (RX). The splitting ratio is 1:1, which is used to generate and receive terahertz waves. When the terahertz waves are focused on the sample surface and scattered, they are collected by the focusing test system. The focal length of the focusing parabolic mirror is 16 mm. The collected signal is amplified by a current amplifier and then connected to the input terminal of the lock-in amplifier. The current amplification factor is 20 MV / cm. The probe jitter frequency in the terahertz near-field microscope is xΩ, and x can be obtained according to the actual situation. This frequency is input into the near-field microscope controller and demodulated through 4 channels, respectively demodulating the frequency signals of 0Ω, 1Ω, 2Ω, and 3Ω. Here, 0 represents non-demodulation, that is, the topography imaging channel of the atomic force microscope itself. 1, 2, and 3 respectively represent three different orders of near-field imaging signals. The higher the order, the weaker the signal. Therefore, generally, the strongest first three orders of near-field signals are taken for imaging. Of course, other orders can also be set for imaging. The demodulated frequency is connected to the lock-in amplifier. The lock-in amplifier inputs the four-order demodulated signals of 0Ω, 1Ω, 2Ω, and 3Ω into the computer, and frequency demodulation imaging is performed on the signals of these 4 channels respectively. The near-field information reflected by the signals of different orders is different. Generally speaking, the higher-order signals can reflect the essence of the near-field information more, but the intensity of the higher-order signals will be weaker.

[0096] The specific process of the fluorescence part is as follows: The fluorescent mercury lamp passes through the filter and then is focused on the sample through the objective lens. The focusing multiples are 4X, 10X, 50X, and 100X respectively. After irradiating the sample surface to generate fluorescence, the fluorescence returns through the objective lens optical path and finally reaches the fluorescent CCD array surface for receiving imaging.

[0097] The above process can perform non-destructive super-resolution near-field imaging and fluorescence imaging on biomedical samples such as cells, viruses, biological macromolecules, and biological tissues. The two are simultaneous in-situ measurements. And the near-field imaging can perform high-order demodulation imaging, supporting up to 4-channel imaging at most, and the demodulation order can be input according to actual needs.

[0098] The specific steps of the sample test part are as follows:

[0099] 1. First, turn on the laser. The laser beam is split into transmitted light and received light by a 1:1 beam splitter. The transmitted light passes through the fiber optic delay line and reaches the transmitting antenna to generate terahertz waves, and the received light reaches the receiving antenna through the mirror to receive terahertz waves.

[0100] 2. Turn on the atomic force microscope and adjust the quadrant laser so that it is reflected by the atomic force probe cantilever to the exact center of the quadrant receiver. At this time, then operate the probe to move in (move in the direction close to the sample) until it touches the sample surface.

[0101] 3. At this time, adjust the emission of terahertz waves, which are focused by the first parabolic mirror to the tip of the atomic force probe, so that the terahertz waves are focused between the tip of the atomic force probe and the sample. The output electrical signal of the receiving antenna is added to a current amplifier and then connected to the input terminal of a lock-in amplifier through the current amplifier.

[0102] 4. Turn on the lock-in amplifier and set the demodulation frequencies of the 4 channels to demodulate the frequency signals of 0Ω, 1Ω, 2Ω, and 3Ω respectively. The corresponding imaging channels are respectively connected to the four channels of the atomic force microscope, and the final output is atomic force topography imaging, first-order near-field imaging, second-order near-field imaging, and third-order near-field imaging. This step is set in the lock-in software at the processor (such as a computer) end. After the setting is completed, the terahertz near-field test part is ready.

[0103] 5. Turn on the inverted fluorescence microscope part, including the mercury lamp light source and the illumination light source, and turn on the observation camera.

[0104] 6. Switch the objective lens group, select a suitable objective lens to observe the sample first. Generally, first use a low-power objective lens to look, find a suitable area, and then switch to a high-power objective lens to look, and it is necessary to find the position of the sample corresponding to the tip of the atomic force probe.

[0105] 7. After finding the position, switch to the highest-magnification fluorescence objective lens, carefully find the position of the sample corresponding to the tip of the probe, and take a fluorescence image of this point after finding it.

[0106] 8. After taking the fluorescence image at the tip of the probe, switch the high-power objective lens to the lowest-power objective lens. At this time, start scanning the terahertz near-field image of the area at this point. Because the terahertz near-field scanning is performed in a very small area of the sample, the sample stage will perform three-dimensional movements. To ensure the safety of the atomic force tip and the highest-magnification objective lens, the objective lens here must be switched to the lowest magnification to ensure a certain spatial distance from the sample.

[0107] The sample replacement process is as Figure 3 shown, Figure 3 which is a schematic diagram of the sample replacement method of the display mirror in the present invention. Lower the fluorescence focusing objective lens by a distance of 10 mm through the displacement stage system, and then lower the sample stage by about 5 mm or so to make it away from the probe tip system. The order of these two steps cannot be changed, and the descending distance of the former must be greater than that of the latter to avoid damage to the objective lens and the probe during the process. Then use tweezers to retrieve the old sample and place the new sample. After placing, first perform the needle insertion operation with the sample stage to make the surface of the sample reach the tip, and then perform the focusing operation of the objective lens. The order of the steps in this process cannot be changed either to ensure that near-field imaging and fluorescence imaging can be performed simultaneously.

[0108] Among them, the hollow sample stage in the present invention is also an atomic force scanning stage. There are a total of 4 fluorescence objective lens groups with magnification factors of 4 times, 10 times, 50 times, and 100 times respectively, which are used to observe the fluorescence of the sample. The microscope also includes an objective turret, which is convenient for switching objective lenses with different magnification factors for observation. The microscope in the present invention also includes a sample clamp, which is located between the atomic force tip and the focal point of the objective lens and is integrally fixed on the hollow sample stage.

[0109] The present invention realizes in-situ real-time imaging of cells or biological tissues. In-situ real-time imaging measurement means that compared with previous measurements, if one wants to simultaneously measure the terahertz near-field information and fluorescence information of the same point on the sample, the general method is to first measure with a fluorescence microscope, and then take the sample to the terahertz near-field microscope to find the same area and then measure; this basically takes several hours because it takes time to adjust the near-field microscope; if it is some sensitive biological samples, the sample has become invalid during this time; moreover, it is not easy to find the same point of the sample between different microscopes, which requires high experience of the operator. The terahertz near-field fluorescence combined microscope can directly measure the near-field measurement and fluorescence measurement of the same point on the sample. One can directly measure the near-field signal by lowering the needle while measuring the fluorescence, truly achieving in-situ (the same position) real-time imaging measurement.

[0110] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0111] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A terahertz near-field microscope that can be used in combination with fluorescence, characterized in that, the terahertz near-field microscope includes: a terahertz unit, an atomic force microscope unit, a fluorescence microscope unit, and a processing unit, where: The terahertz unit includes: a femtosecond laser light source, an optical fiber beam splitter, an optical fiber delay line, a transmitting antenna, a first focusing parabolic mirror, a second focusing parabolic mirror, and a receiving antenna; the femtosecond laser light source is used to generate laser light, which is divided into a first transmitted light and a first received light by the optical fiber beam splitter. The first transmitted light is delayed by the optical fiber delay line and then focused on the transmitting antenna to generate terahertz waves. The optical fiber delay line can also be used to adjust the distance between the transmitting antenna and the receiving antenna to match the optical path of the fluorescence objective lens body. The terahertz waves are focused by the first focusing parabolic mirror into the region between the atomic force probe tip and the sample, generating scattered terahertz near-field signals. The terahertz near-field signals are focused by the second focusing parabolic mirror and then emitted collinearly and focused on the receiving antenna. The terahertz near-field signals and the first received light act on the receiving antenna to cause the carriers to move directionally and generate a first electrical signal. The first electrical signal is transmitted to the processing unit and obtained as the terahertz near-field spectral image of the sample after being processed by phase-locked demodulation; The atomic force microscope unit includes: an atomic force probe, a quadrant laser emitter, a quadrant detector, and a sample stage; the quadrant laser emitter is used to emit laser light to the tip cantilever of the atomic force probe. The laser light is reflected back to the quadrant detector by the tip cantilever. The sample stage is a hollow stage, and the sample is suspended in the hollow area of the sample stage; The fluorescence microscope unit includes: a high-pressure mercury lamp, an excitation filter, a first dichroic mirror, a fluorescence objective lens, an illumination light source, a collimating lens, a second dichroic mirror, an emission filter, and a fluorescence camera; The illumination light source is used to emit illumination light. The illumination light is collimated by the collimating lens and then enters the second dichroic mirror. The illumination light is reflected by the second dichroic mirror and then enters the fluorescence objective lens. The illumination light is focused by the fluorescence objective lens and then irradiates the sample on the sample stage to illuminate the sample surface; the high-pressure mercury lamp is used to generate radiation light in the ultraviolet to infrared band. The radiation light is filtered by the excitation filter to generate a preset wavelength characteristic light source. The preset wavelength characteristic light source is reflected by the first dichroic mirror and then enters the fluorescence objective lens and is focused on the sample on the sample stage to excite fluorescence. The fluorescence is reflected and then passes through the fluorescence objective lens, the first dichroic mirror, the second dichroic mirror, and the emission filter in sequence and then enters the fluorescence camera. The fluorescence camera transmits the collected fluorescence data information to the processing unit, and the processing unit processes the information collected based on the fluorescence camera and obtains the fluorescence image of the sample.

2. A terahertz near-field microscope that can be used in combination with fluorescence according to claim 1, characterized in that, the atomic force microscope unit further includes at least one support member and a housing. The lower end of the support member is connected to the sample stage, the upper end of the support member is connected to the lower end of the housing. The quadrant laser emitter and the quadrant detector are located inside the housing. The housing is respectively provided with holes for adjusting the laser light emitted by the quadrant laser emitter and for adjusting the laser light received by the quadrant detector.

3. A terahertz near-field microscope that can be used in combination with fluorescence according to claim 2, characterized in that, The atomic force microscope unit also includes a first observation camera, which is used to obtain the real-time position of the atomic force probe. A hole for observation by the first observation camera is opened on the top of the shell; the height of the support member is respectively greater than the height of the first focusing parabolic mirror and the height of the second focusing parabolic mirror.

4. The terahertz near-field microscope that can be used in conjunction with fluorescence according to claim 1, It is characterized in that The terahertz near-field microscope also includes a current amplifier and a lock-in amplifier. The first electrical signal is input into the current amplifier for amplification, the amplified signal is input into the lock-in amplifier for demodulation, and the signal demodulated by the lock-in amplifier is input into the processing unit.

5. A terahertz near-field microscope that can be used in conjunction with fluorescence according to claim 4, It is characterized in that The demodulation frequencies of four signal channels are set in the phase-locked amplifier. The four signal channels correspond to four imaging channels respectively. The four imaging channels correspond to output atomic force morphology imaging, first-order near-field imaging, second-order near-field imaging and third-order near-field imaging respectively.

6. The terahertz near-field microscope that can be used in conjunction with fluorescence according to claim 1, It is characterized in that The terahertz unit is located above the sample stage, and the fluorescence microscope unit is located below the sample stage.

7. The terahertz near-field microscope that can be used in conjunction with fluorescence according to claim 1, It is characterized in that The sample stage is provided with a movement control unit for controlling the movement of the sample stage in three directions: X-axis, Y-axis and Z-axis.

8. A method for using a terahertz near-field microscope that can be used in conjunction with fluorescence according to claim 4, It is characterized in that The method comprises: Test steps: Step 1: Suspend the test sample in the hollow area of ​​the sample stage, and place the sample on the substrate; Step 2: Turn on the femtosecond laser light source; Step 3: Turn on the four-quadrant laser emitter and adjust the four-quadrant laser emitter so that the laser emitted by the four-quadrant laser emitter is reflected back to the center of the four-quadrant detector through the needle tip cantilever, and then the atomic force probe is inserted until it contacts the surface of the test sample; Step 4: Adjust the transmitting antenna so that the terahertz wave is focused to the tip of the atomic force probe through the first focusing parabolic mirror, and the receiving antenna outputs the first electrical signal to the current amplifier, which is amplified by the current amplifier and input to the input end of the phase-locked amplifier; Step 5: Turn on the lock-in amplifier, set the demodulation frequency of the four signal channels in the lock-in amplifier, and connect the four signal channels to the corresponding four imaging channels. The four imaging channels correspond to the output of atomic force topography imaging, first-order near-field imaging, second-order near-field imaging, and third-order near-field imaging respectively; Step 6: Turn on the high-pressure mercury lamp, the illumination light source and the fluorescent camera; Step 7: Use a fluorescent objective lens to find the test sample position corresponding to the atomic force probe tip, and then take a fluorescent image of the position; Step 8: After taking the fluorescence image, switch the objective lens group in the fluorescence objective lens from high magnification to low magnification, thus completing the test steps; Measurement steps: After the test step is completed, the test sample is replaced with the test piece for measurement.

9. The terahertz near-field microscope that can be used in conjunction with fluorescence according to claim 8, It is characterized in that Step 7 includes: Step 7.1: Select the low-magnification objective lens group in the objective lens group of the fluorescence objective lens to preliminarily observe and obtain the sample position range corresponding to the atomic force probe tip; Step 7.2: Based on this range, switch the low-magnification objective lens group in the fluorescence objective lens to the high-magnification objective lens group, find the sample position corresponding to the atomic force probe tip, and then take a fluorescence image of this position.

10. A terahertz near-field microscope that can be used in combination with fluorescence according to claim 8, characterized in that, the method further includes: a replacement step for the new sample to be measured: Step a: Lower the fluorescence objective lens by a first distance value; Step b: Lower the sample stage by a second distance value, where the first distance value is greater than the second distance value; Step c: Take out the original sample in the sample stage and put the new sample into the sample stage.

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