Microscopic imaging system with resolution exceeding light spot size limitation

Abstract

The invention relates to the technical field of microscopic imaging, and discloses a microscopic imaging system with resolution exceeding light spot size limitation. The system comprises a laser unit; the measured target unit is arranged in the reference arm light path; the tested target unit comprises a tested sample and a divergence angle adjusting element; the divergence angle adjusting element is used for adjusting the divergence angle of the reference light source passing through the tested sample; the spatial radio frequency marking unit comprises a reference arm light path and a measuring arm light path and is used for performing optical heterodyne interference processing on the laser beams according to the radio frequency driving signals of various frequencies to form a reference light source and a light spot array respectively; the reference light source enters the measured target unit and then is combined with the light spot array to generate a combined light beam; the image signal receiving unit is used for collecting the combined light beam and then generating light data; and the data processing unit is used for receiving the optical data and carrying out image inversion processing on the optical data to generate imaging data of the tested sample. According to the scheme, the resolution limit of the system is improved.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging technology, and in particular to a microscopic imaging system with a resolution exceeding the limit of spot size. Background Art

[0002] Laser scanning imaging generates high-resolution images by scanning a sample with a laser beam. Spatial radio frequency tagging uses radio frequency signals to label specific molecules or structures, providing molecular-level information. Microscopic imaging technology combining laser scanning imaging with spatial radio frequency tagging offers advantages in high resolution and molecular specificity, and has important applications in biomedical research. The resolution limit of microscopic imaging based on spatial tagging, like other laser scanning microscopy techniques, is determined by the numerical aperture of the devices in the optical path and the laser spot size limited by the laser wavelength.

[0003] In related technologies, the smallest resolvable spot size that can be achieved by an actual optical system is the system resolution limit. Due to the non-ideality of optical system design and light source components, the actual spot size is often larger than the ideal spot size, resulting in a deterioration in system resolution. Furthermore, due to limitations in processing technology and optical paths, it is difficult to further reduce the laser spot size to improve resolution. Forcibly setting up related devices will increase equipment costs and limit system performance.

[0004] Therefore, there is an urgent need for a microscopic imaging solution that can improve the system resolution and surpass the laser scanning imaging resolution under the premise of a certain system spot size. Summary of the Invention

[0005] In view of this, the present invention provides a microscopic imaging system with a resolution exceeding the spot size limit to solve the problem of limited microscopic imaging resolution. The technical solution is as follows:

[0006] A microscopic imaging system with a resolution exceeding the spot size limit is provided, the system comprising:

[0007] a laser unit, for generating a laser beam;

[0008] A target unit is provided in the optical path of the reference arm; the target unit includes a sample to be measured and a divergence angle adjustment element; the divergence angle adjustment element is used to adjust the divergence angle of the reference light source passing through the sample to be measured;

[0009] A spatial radio frequency tag unit includes a reference arm optical path and a measurement arm optical path, which are used to perform optical heterodyne interference processing on the laser beam according to radio frequency drive signals of multiple frequencies to form a reference light source and a light spot array, respectively; after the reference light source is incident on the measured target unit, it is combined with the light spot array to generate a combined light beam; different light spots in the light spot array correspond to different light frequencies;

[0010] an image signal receiving unit, configured to collect the combined light beam and generate light data;

[0011] The data processing unit is used to receive the light data and perform image inversion processing on the light data to generate imaging data of the measured sample.

[0012] In an optional embodiment, the divergence angle adjustment element is a continuous zoom element.

[0013] In an optional embodiment, the spatial radio frequency tag unit also includes a radio frequency driver, a first spatial light modulator and a second spatial light modulator; the radio frequency driver is used to send radio frequency drive signals to the first spatial light modulator and the second spatial light modulator respectively; the first spatial light modulator is used to perform heterodyne interference processing on the laser light beam according to the received radio frequency drive signal to form a reference light source; the second spatial light modulator is used to perform heterodyne interference processing on the laser light beam according to the received radio frequency drive signal to form a light spot array.

[0014] In an optional embodiment, the reference arm optical path also includes a beam shaping device and a first beam reduction element; the beam shaping device is used to shape the laser beam processed by the first spatial light modulator into a reference light source with uniform light intensity; the first beam reduction element is used to reduce the reference light source and then inject it into the target unit to be measured.

[0015] In an optional embodiment, the spatial radio frequency tag unit further includes a beam splitter and a beam combiner;

[0016] The beam splitter is used to split the laser beam into a first laser beam and a second laser beam; the first laser beam is incident on the reference arm optical path; the second laser beam is incident on the measurement arm optical path;

[0017] The beam combiner is used to combine the outgoing light beam after entering the target unit to be measured with the light spot array to generate a combined light beam.

[0018] In an optional embodiment, the measured target unit further includes a displacement device; the measured sample is fixed on the displacement device; and the displacement device is used to drive the measured sample to move to achieve push-scan imaging.

[0019] In an optional embodiment, the spatial radio frequency tag unit further includes a second beam reduction element; the image signal receiving unit includes a unit point detector;

[0020] The second beam-shrinking element is used to shrink the combined light beam so as to be incident on the unit point detector; the unit point detector is used to perform photoelectric conversion on the incident light beam to generate optical data.

[0021] In an optional embodiment, the data processing unit includes an acquisition card and an image inversion unit; the acquisition card is used to acquire the light data and perform analog-to-digital conversion to generate digital light data; the image inversion unit is used to convert the digital light data into the frequency domain to perform image inversion processing to generate imaging data of the sample under test.

[0022] In an optional embodiment, the laser unit includes a plurality of lasers; the plurality of lasers respectively generate laser beams of different wavelength bands;

[0023] The image signal receiving unit includes a plurality of unit point detectors; the plurality of unit point detectors correspond one-to-one to the plurality of lasers and are used to respectively collect corresponding wavelength bands of the combined light beam to generate corresponding light data.

[0024] In an optional embodiment, the image signal receiving unit further includes a plurality of filters; the plurality of filters correspond one-to-one to the plurality of unit point detectors, and are used to filter the combined light beams into corresponding wavelength bands and then input them into corresponding unit point detectors.

[0025] In an optional embodiment, the system further includes a host computer for visually displaying the imaging data.

[0026] The technical solution provided by the present invention can have the following beneficial effects:

[0027] The present invention provides a microscopic imaging system with a resolution exceeding the limit of the spot size, comprising a laser unit, a target unit to be measured, a spatial radio frequency tag unit, an image signal receiving unit and a data processing unit. The laser unit is used to generate a laser beam. The target unit to be measured is arranged in the reference arm optical path, and comprises a sample to be measured and a divergence angle adjustment element, and the divergence angle adjustment element is used to adjust the divergence angle of the reference light source passing through the sample to be measured. The spatial radio frequency tag unit comprises a reference arm optical path and a measuring arm optical path, which are used to perform optical heterodyne interference processing on the laser beam according to radio frequency driving signals of multiple frequencies, respectively, to form a reference light source and a spot array, respectively. After the reference light source is incident on the target unit to be measured, it is combined with the spot array to generate a combined light beam. Different spots in the spot array correspond to different light frequencies. The image signal receiving unit is used to generate light data after collecting the combined light beam. The data processing unit is used to receive the light data, and perform image inversion processing on the light data to generate imaging data of the sample to be measured. By placing the target unit under test in the reference arm optical path and setting a divergence angle adjustment element to adjust the divergence angle of the reference light source passing through the sample under test, the system resolution can be adjusted. This breaks through the system's inherent situation where the resolution limit is determined by the spot size, greatly improving the system's resolution limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 2 is a schematic structural diagram of a microscopic imaging system with a resolution exceeding the spot size limitation according to an embodiment of the present invention;

[0030] Figure 2 is a schematic structural diagram of another microscopic imaging system with a resolution exceeding the spot size limitation according to an embodiment of the present invention;

[0031] Figure 3 is a schematic diagram of imaging results according to an embodiment of the present invention;

[0032] Figure 4 2 is a schematic structural diagram of a microscopic imaging system with a resolution exceeding the spot size limitation according to an embodiment of the present invention;

[0033] Figure 5 2 is a schematic structural diagram of a multi-channel microscopic imaging system with a resolution exceeding the spot size limitation according to an embodiment of the present invention;

[0034] Figure 6 3 is a schematic structural diagram of a microscopic imaging system according to an embodiment of the present invention, wherein the resolution exceeds the limit of the spot size and is continuously adjustable.

[0035] Description of reference numerals:

[0036] 1-laser unit; 2-first glass slide; 3-second glass slide; 4-third glass slide; 5-fourth glass slide; 6-fifth glass slide; 7-beam splitter; 8-beam combiner; 9-first spatial light modulator; 10-second spatial light modulator; 11-first reflector; 12-second reflector; 13-beam expander; 14-objective lens; 15-tested sample; 16-lens; 17-unit point detector; 18-data processing unit; 19-host computer; 20-RF driver; 21-first convex lens; 22-second convex lens; 23-aperture; 24-beam homogenizer; 25-dichroic mirror group; 26-lens group; 27-filter group; 28-multi-channel unit point detector; 29-continuous zoom element. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0038] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0039] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0040] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0041] Figure 1 FIG. 1 is a schematic diagram of a microscopic imaging system according to an embodiment of the present invention, wherein the resolution exceeds the limit of the spot size. Figure 1 As shown, the microscopic imaging system with a resolution exceeding the spot size limit includes a laser unit, a target unit, a spatial radio frequency tag unit, an image signal receiving unit, and a data processing unit.

[0042] A laser unit is used to generate a laser beam.

[0043] The measured target unit is arranged in the reference arm optical path; the measured target unit includes a measured sample and a divergence angle adjustment element; the divergence angle adjustment element is used to adjust the divergence angle of the reference light source passing through the measured sample.

[0044] The spatial radio frequency tag unit includes a reference arm optical path and a measurement arm optical path, which are used to perform optical heterodyne interference processing on the laser beam according to radio frequency driving signals of multiple frequencies to form a reference light source and a light spot array respectively; after the reference light source is incident on the target unit to be measured, it is combined with the light spot array to generate a combined beam; different light spots in the light spot array correspond to different light frequencies.

[0045] The image signal receiving unit is used to collect the combined light beam and generate light data.

[0046] The data processing unit is used to receive the light data and perform image inversion processing on the light data to generate imaging data of the measured sample.

[0047] Figure 1 The working principle of the microscopic imaging system that shows the resolution beyond the spot size limit is as follows:

[0048] The laser unit is used to generate a laser beam, serving as the system's light source. After entering the optical path and being modulated or shaped, the laser beam illuminates the sample under test, generating an image signal. Optionally, the laser unit includes a laser with a narrow linewidth and high stability. The resulting spot shape can be circular, elliptical, rectangular, or other shapes, with different initial resolutions corresponding to different shapes.

[0049] The spatial radio frequency tagging unit is used to implement spatial radio frequency tagging technology, which is a technology that uses frequency signals to mark spatial position coordinates. It is implemented by using heterodyne interference in the optical field, such as Mach-Zehnder interference and other interference optical paths. Specifically, the heterodyne interference optical path includes a reference arm optical path and a measuring arm optical path, which divides the laser beam into two paths, entering the reference arm optical path and the measuring arm optical path respectively. The reference arm optical path realizes the shaping of the incident corresponding laser beam, and obtains a reference light source to be incident on the target unit to be measured. The incident light is irradiated on the measured sample. The measured sample has different transmission rates for light at different spatial positions, causing the light intensity at the corresponding position to attenuate, thereby weakening the frequency domain signal amplitude at the corresponding position after the subsequent beat, affecting the subsequent generated image of the measured sample; the measuring arm optical path realizes the spatial modulation of the incident corresponding laser beam, introducing a frequency shift related to the driving frequency of the radio frequency driving signal to the laser spot of the laser beam, and modulating the laser spot into a one-dimensional or two-dimensional spot array. The spot spacing and the number of spots in the spot array are related to the radio frequency driving signal. Finally, the reference light source of the target unit to be measured is combined with the light spot array to generate a beat frequency signal, realizing heterodyne interference of the one-dimensional or two-dimensional light spot array. After heterodyne interference, each light spot in the light spot array has a specific beat frequency envelope, and each beat frequency envelope has a specific frequency, realizing the marking of the spatial position of the light spot by using the beat frequency.

[0050] Since the reference light source of the sample under test in the target unit is divergent light, a divergence angle adjustment element is also provided in the target unit to adjust the divergence angle of the reference light source passing through the sample under test. The divergence ability of the divergence angle adjustment element to light is the key to determining the system resolution. By adjusting the divergence angle of the divergence angle adjustment element, the system resolution can be adjusted. The smaller the divergence angle, the smaller the spot size, the more concentrated the energy, and the higher the system resolution, thereby achieving microscopic imaging with a resolution beyond the spot size limit.

[0051] The image signal receiving unit is used to collect the beat frequency light signal in the combined light beam, and convert the collected light signal into an electrical signal. After obtaining the light data, it is input into the data processing unit. The light data is a time-domain voltage signal with optical information. The data processing unit receives the light data output by the image signal receiving unit, and runs the image inversion algorithm to process the light data, realize image inversion, and obtain imaging data of the sample under test.

[0052] Optionally, the divergence angle adjustment element can be a combination of one or more optical devices. If a fixed-focus element is used as the divergence angle adjustment element, the system resolution is a resolution corresponding to the fixed-focus element. By using a combination of optical devices with different focal lengths as the divergence angle adjustment element and adjusting them as needed, the system resolution can be modified.

[0053] Optionally, the divergence angle adjustment element is a convex lens or a concave lens. By adjusting the focal length of the convex lens or the concave lens, the divergence angle of the passing laser beam can be adjusted, thereby adjusting the system resolution.

[0054] In an optional embodiment, the divergence angle adjustment element is a continuous zoom element. By using the continuous zoom element as the divergence angle adjustment element, the divergence angle of the passing laser light beam can be continuously adjusted, and then the magnification of the microscopic image can be continuously adjusted accordingly, thereby achieving continuous adjustment of the system resolution, improving the adjustment accuracy, and thus improving the accuracy of microscopic imaging.

[0055] In an optional embodiment, the spatial radio frequency tag unit further includes a radio frequency driver, a first spatial light modulator, and a second spatial light modulator. The radio frequency driver is configured to send radio frequency drive signals to the first spatial light modulator and the second spatial light modulator, respectively. The first spatial light modulator is configured to perform heterodyne interferometry on the laser beam based on the received radio frequency drive signal to form a reference light source. The second spatial light modulator is configured to perform heterodyne interferometry on the laser beam based on the received radio frequency drive signal to form a light spot array.

[0056] A spatial light modulator (SLM) is a device that modulates the spatial distribution of light waves. Under the control of an electrical drive signal, it changes the frequency, amplitude or intensity, phase, polarization state, and other aspects of the spatial light distribution. SLMs include acousto-optic deflectors (AODs), acousto-optic frequency shifters (AOFSs), electro-optic modulators (EOMs), and acousto-optic modulators (AOMs). The input to the SLM is an RF drive signal. RF drive signals of different frequencies drive the SLM to produce different modulation effects. By applying RF drive signals of different frequencies to the SLM through an RF driver, different parameters can be controlled. The spacing and number of spots in the modulated light spot array are related to the spacing and number of the frequency components of the RF drive signal applied to the second SLM. A single-frequency or multi-frequency composite RF drive signal can also be input. The multi-frequency composite RF drive signal is loaded on the spatial light modulator and can be regarded as the superposition of the individual effects of each single-frequency RF drive signal contained in the multi-frequency composite RF drive signal at the same time. The more frequencies there are in the composite RF drive signal, the worse the effect of each frequency.

[0057] Exemplarily, an acousto-optic deflector (AOD) is used as a spatial light modulator to apply a certain frequency shift to the incident light. The magnitude of the frequency shift is the same as the frequency of the RF drive signal applied to the acousto-optic deflector (AOD), and the outgoing light has a certain deflection angle relative to the incident light. The magnitude of the deflection angle is related to the frequency of the RF drive signal applied to the acousto-optic deflector (AOD). When a single acousto-optic deflector (AOD) is used, a single-frequency RF drive signal can be applied to the acousto-optic deflector (AOD) in sequence to achieve point scanning; or, a multi-frequency composite RF drive signal can be applied to the acousto-optic deflector (AOD) at the same time to generate a one-dimensional light spot array. When two orthogonally placed acousto-optic deflectors (AODs) are used, a multi-frequency composite RF drive signal can be applied to each of the two acousto-optic deflectors (AODs) at the same time to generate a two-dimensional light spot array.

[0058] Optionally, the first spatial light modulator may be replaced by a spatial light modulator group consisting of a plurality of spatial light modulators, so that the emitted reference light source has a fixed frequency shift.

[0059] Optionally, the second spatial light modulator can also be replaced by a spatial light modulator group consisting of multiple spatial light modulators, for example, a spatial light modulator group consisting of an orthogonally placed third spatial light modulator and a fourth spatial light modulator to modulate the laser spot of the incident second laser beam into a two-dimensional spot array.

[0060] In an optional embodiment, the reference arm optical path also includes a beam shaping device and a first beam reduction element; the beam shaping device is used to shape the laser beam processed by the first spatial light modulator into a reference light source with uniform light intensity; the first beam reduction element is used to reduce the reference light source and then inject it into the target unit to be measured, so as to converge the reference light source to the test area of the sample to be measured, and ensure that the energy of the light is concentrated in the test area as much as possible, so as to make full use of the energy of the light. The reference light source is a two-dimensional surface light source. The shaped reference light source must be able to cover the light spot array to ensure that each light spot can achieve heterodyne interference. After heterodyne interference, each light spot in the light spot array has a specific beat frequency envelope, and each beat frequency envelope has a specific frequency, so that the beat frequency can be used to mark the spatial position of the light spot.

[0061] Optionally, the first beam-reducing element is a convex lens.

[0062] Optionally, the beam shaping device includes a beam expander and an aperture. The beam expander is used to amplify the laser spot of the laser beam processed by the first spatial light modulator, so that the relatively concentrated and flat laser spot after amplification can be used as a reference light source. The aperture is used to adjust the light flux of the laser beam passing through the beam expander, optimize imaging quality, and perform shaping and filtering. Together with the beam expander, it shapes the incident laser beam to obtain a reference light source with uniform light intensity. Powell prisms, cylindrical lenses, commercial beam shapers, beam homogenizers, etc. can also be used as beam shaping devices to achieve shaping of the incident laser beam.

[0063] In an optional embodiment, the spatial radio frequency tag unit further includes a beam splitter and a beam combiner. The beam splitter is configured to split the laser beam into a first laser beam and a second laser beam, wherein the first laser beam enters the reference arm optical path and the second laser beam enters the measurement arm optical path. The beam combiner is configured to combine the outgoing beam after entering the target unit with the light spot array to generate a beat frequency and thus form a combined beam.

[0064] Optionally, a first glass slide is further provided between the laser unit and the beam splitter. The first glass slide is a 1 / 2 glass slide. The laser beam generated by the laser unit has different polarization states, and the beam splitter is a polarization beam splitter. The laser beam emitted by the laser unit first passes through the first glass slide and the polarization beam splitter before being split into two beams of polarization states perpendicular to each other: P light (parallel-polarized light, corresponding to the first laser beam) and S light (perpendicular-polarized light, corresponding to the second laser beam). By rotating the first glass slide, the proportion of the first laser beam and the second laser beam to the laser beam can be changed to adjust the light intensity of the first laser beam and the light intensity of the second laser beam.

[0065] In an optional embodiment, the measured target unit further includes a displacement device, the measured sample is fixed on the displacement device, and the displacement device is used to drive the measured sample to move to achieve push-scan imaging.

[0066] Optionally, the displacement device further includes a clamping structure for securing the sample under test. The displacement device can drive the sample under test to perform multi-axis movement, achieving high-precision, high-stability driving of the sample under test. When the spot array is a one-dimensional array, one-dimensional linear array push-scanning and swing-scanning imaging can be achieved. When the spot array is a two-dimensional array, two-dimensional area array push-scanning and swing-scanning imaging can be achieved. The sample under test can be moved perpendicular to the optical path between the beam shaping device and the beam combiner, achieving splicing imaging of samples with larger test surfaces. Exemplarily, the displacement device is a motorized translation stage.

[0067] Alternatively, if the sample being measured has uniaxial or multi-axis motion capabilities, such as a flow cytometer, there's no need to add a displacement device. The flow cytometer's optical path can be placed directly on the target area being measured, with the flow axis acting as the displacement axis to achieve push-scan imaging. If the laser spot in the measurement arm of the heterodyne interferometer optical path is modulated into a two-dimensional spot array, and the laser spot in the reference arm is shaped into a surface light source, video imaging of dynamic scenes can be achieved directly without adding a displacement device.

[0068] In an optional embodiment, the spatial radio frequency tag unit further includes a second beam reduction element. The image signal receiving unit includes a unit point detector. The second beam reduction element is used to reduce the combined beam to a size that can be incident on the target surface of the unit point detector, and ensure that the focus of the measured sample pattern formed by the light spot array of the measurement arm optical path and the reference light source of the reference arm optical path after passing through the measured sample is on the unit point detector, so as to be incident on the unit point detector. The unit point detector is used to receive the incident light beam to obtain a time-domain optical signal, perform photoelectric conversion processing on the time-domain optical signal obtained based on the incident light beam, and generate optical data converted into an electrical signal. The unit point detector is a non-imaging unit detector that can only sense the intensity of the light signal. It is widely used in fields such as confocal, two-photon, and single-molecule imaging. Exemplary examples of the unit point detector include a photomultiplier tube (PMT), a photodiode (PD), an avalanche photodiode (APD), and a single-photon avalanche diode (SPAD). Different unit point detectors can be selected as needed for different samples to be measured. Compared to commonly used imaging detectors (such as charge-coupled devices (CCDs) and complementary metal-oxide-semiconductors (CMOSs), single-point detectors offer the advantages of high detection sensitivity and high frame rates, offering significant speed and sensitivity advantages in microscopic imaging applications. The time-domain signal of a single-point detector is the intensity signal of the optical beat frequency signal generated by the heterodyne interference light path passing through the sample being measured. It is a superposition of multi-frequency, multi-amplitude signals, representing the time-varying sum of the power of the light signal incident on the detector target surface.

[0069] Optionally, the second beam-reducing element is an objective lens; or, the second beam-reducing element is a lens.

[0070] Optionally, by adjusting the distance between the divergence angle adjustment element and the sample to be measured, the imaging focus position of the sample to be measured can be adjusted after beam combining, and then the relative position of the imaging focus position and the target surface of the unit point detector can be adjusted to ensure that the light beams of the reference arm optical path and the measuring arm optical path are focused at the same target position after beam combining, and the target surface of the unit point detector is placed at this target position.

[0071] In an optional embodiment, the data processing unit includes an acquisition card and an image inversion unit. The optical data output by the image signal receiving unit is a time-domain voltage signal carrying optical information, which is an analog signal. The acquisition card is used to acquire the optical data and perform analog-to-digital conversion to generate digital optical data. The image inversion unit is used to receive the digital optical data output by the acquisition card, convert the digital optical data into the frequency domain, calculate the amplitude of a specific frequency point, and each frequency point corresponds to a spatial coordinate to perform image inversion processing. The frequency is equal to the coordinate position, and the frequency peak is equal to the signal intensity, thereby generating imaging data of the sample under test. Specifically, the image inversion unit obtains the frequency domain distribution of the signal by analyzing the time domain information in the digital optical data, and uses the amplitude intensity of the specific frequency point of the frequency domain signal to perform intensity inversion on the optical signal at the corresponding spatial position, inverting the frequency domain assignment of the one-dimensional spot array into one-dimensional image data, and inverting the frequency domain assignment of the two-dimensional spot array into two-dimensional image data. Optionally, the time domain signal is converted into a frequency domain signal by Fourier transform.

[0072] Optionally, the acquisition card is a high-speed data acquisition board with a sampling rate at least greater than or equal to twice the maximum frequency of the beat frequency envelope, and a bandwidth covering all envelope frequencies. The image inversion unit can be implemented on a high-speed hardware platform, such as a field programmable gate array FPGA (Field Programmable Gate Array), ZYNQ (a fully programmable system on chip), a central processing unit CPU (Central Processing Unit), a graphics processing unit GPU (Graphics Processing Unit) and other high-speed data processing devices to perform real-time, high-throughput image inversion. Exemplarily, the image inversion unit runs a Fourier transform algorithm on a high-speed hardware platform to convert the time domain signal from the unit point detector collected by the acquisition card into a frequency domain signal. Each beat frequency envelope frequency corresponds to a frequency point, corresponds to a specific light spot, represents a specific spatial position of a sample under test, that is, a pixel coordinate in the image. The peak intensity of the frequency is the signal intensity of the point, representing the transmittance of the sample under test at that point. The frequency value and the peak intensity at the frequency value are used to realize the inversion of the image coordinates and pixel values to achieve imaging.

[0073] In an optional embodiment, the laser unit includes multiple lasers; each of the multiple lasers generates a laser beam of a different wavelength band. For different samples to be measured, one or more lasers of different wavelength bands can be selected as required to generate corresponding laser beams that are input into the optical path. Accordingly, when multiple lasers are provided in the laser unit so that multiple laser wavelength bands are incident simultaneously, the image signal receiving unit includes multiple unit point detectors, which correspond one-to-one with the multiple lasers and are used to respectively collect the corresponding wavelength bands of the combined beam to generate corresponding optical data, thereby achieving simultaneous acquisition of multispectral signals.

[0074] In an optional embodiment, the image signal receiving unit also includes multiple filters, which correspond one-to-one to the multiple unit point detectors. The filters only collect spectral signals of corresponding bands to extract the spectral signals of the sample under test. The filters are used to filter the combined light beams to the corresponding bands and then input them into the corresponding unit point detectors.

[0075] In an optional embodiment, the system also includes a host computer for visualizing the imaging data, converting the imaging data into images and displaying them on a screen. The host computer can also serve as a control terminal to implement functions such as control between multiple devices in the system, task scheduling, image display, and command issuance.

[0076] As one or more specific application examples of the embodiments of the present invention, the optimal implementation scheme or the solution that the inventor most wants to embody is described below in combination with specific application scenarios.

[0077] Figure 2 FIG. 1 is a schematic diagram of a microscopic imaging system according to an embodiment of the present invention, wherein the resolution exceeds the limit of the spot size. Figure 2 As shown, in this microscopic imaging system with a resolution exceeding the spot size limit, a laser beam is generated by a laser unit 1. The laser beam is polarized by the first glass slide 2 and then enters the beam splitter 7 to be split into a first laser beam and a second laser beam. The first laser beam changes its polarization state by the second glass slide 3 and then enters the first spatial light modulator 9. The first spatial light modulator 9 modulates the incident corresponding laser beam according to the RF drive signal sent by the RF driver 20 and then emits it. The output laser beam of the first spatial light modulator is reflected by the first reflector 11 to the beam expander 13 for beam expansion, then enters the aperture 23 for shaping, and then enters the first convex lens 21 (corresponding to the first beam reduction element) for beam reduction before entering the sample to be measured 15. The output beam of the sample to be measured 15 passes through the second convex lens 22 (corresponding to the divergence angle adjustment element) to adjust the divergence angle and imaging focus position, and then passes through the fourth glass slide 5 to adjust the polarization state before entering the beam combiner 8; the second laser beam passes through the third glass slide 4 to adjust the polarization state and then is reflected by the second reflector 12 to the second spatial light modulator 10 ( Figure 2The two orthogonally placed spatial light modulators are taken as an example) and then modulated and emitted. The outgoing laser beam of the first spatial light modulator passes through the fifth glass slide 6 to adjust the polarization state and then enters the beam combiner 8. After the two laser beams are combined in the beam combiner 8, they enter the objective lens 14 and the lens 16 (corresponding to the second beam reduction element) in sequence for beam reduction and then enter the unit point detector 17 to generate light data. The data processing unit 18 collects the light data and performs image inversion processing. After the imaging data is generated, it is uploaded to the host computer 19. The host computer 19 can convert the imaging data into an image for visual display. The host computer 19 can also issue instructions to the RF driver and the data processing unit respectively to coordinate the work of the system.

[0078] Among them, the first glass slide 2, the second glass slide 3, the third glass slide 4, the fourth glass slide 5, and the fifth glass slide 6 adjust the polarization state of the passing laser light beam to ensure the polarization state requirements of the spatial light modulator and the interference optical path to ensure maximum interference efficiency. The position and number of the glass slides can be adjusted as needed.

[0079] Among them, the purpose of using the beam expander 13 to expand the incident light spot is to adjust the size of the laser spot so that it meets the requirements of the aperture 23 / beam homogenizer 24 for the size of the incident light plate. The beam shaping module can shape the Gaussian spot into a flat-top light, ensuring high uniformity of the spot energy.

[0080] Optionally, the sample to be tested can be a fixed pattern, and the displacement device can be used to drive the sample to move up and down and left and right to achieve inversion imaging; the sample to be tested can also have the characteristic of movement itself. For example, when using a flow cytometer to perform single-cell analysis, the flow cytometer test tube can be directly fixed on the displacement device. The displacement device is no longer used to drive the tube movement. The flow direction of the flow cytometer is used as the scanning axis. Combined with a two-dimensional area array spot array, video imaging can be achieved.

[0081] Optionally, a second convex lens 22 is used as a divergence angle adjustment element. Using a second convex lens 22 with different focal lengths will change the size of the unit size of the sample being measured incident on the objective lens 14. After the beam is reduced, the target surface of the unit point detector corresponds to a different number of line spots. For example, Figure 3 is a schematic diagram of imaging results according to an embodiment of the present invention, such as Figure 3 As shown in the figure, when a convex lens with a focal length of 10mm is used, the image "H" with a width of 45um on the sample under test corresponds to five 12um (1 / e^2) light spots on the target surface of the unit point detector, realizing a microscopic imaging solution that uses a 12um light spot to achieve an imaging resolution of 9um.

[0082] The microscopic imaging system provided in this embodiment has a resolution exceeding the limit of the spot size, and includes a laser unit, a target unit to be measured, a spatial radio frequency tag unit, an image signal receiving unit, and a data processing unit. The laser unit is used to generate a laser beam. The target unit to be measured is arranged in the reference arm optical path, and includes a sample to be measured and a divergence angle adjustment element, and the divergence angle adjustment element is used to adjust the divergence angle of the reference light source passing through the sample to be measured. The spatial radio frequency tag unit includes a reference arm optical path and a measuring arm optical path, which are used to perform optical heterodyne interference processing on the laser beam according to radio frequency driving signals of multiple frequencies, respectively, to form a reference light source and a spot array, respectively. After the reference light source is incident on the target unit to be measured, it is combined with the spot array to generate a combined light beam. Different spots in the spot array correspond to different light frequencies. The image signal receiving unit is used to generate light data after collecting the combined light beam. The data processing unit is used to receive the light data, and perform image inversion processing on the light data to generate imaging data of the sample to be measured. By placing the target unit under test in the reference arm optical path and setting a divergence angle adjustment element to adjust the divergence angle of the reference light source passing through the sample under test, the system resolution can be adjusted. This breaks through the system's inherent situation where the resolution limit is determined by the spot size, greatly improving the system's resolution limit.

[0083] Figure 4 3 is a schematic structural diagram of a microscopic imaging system with a resolution exceeding the spot size limitation according to an embodiment of the present invention. Figure 4 and Figure 2 The difference is that a beam homogenizing mirror 24 is used to replace the aperture 23 to shape the incident laser beam.

[0084] Figure 5 3 is a schematic structural diagram of a multi-channel microscopic imaging system with a resolution exceeding the spot size limitation according to an embodiment of the present invention. Figure 5 The laser unit 1 includes multiple lasers, which generate laser beams of different wavelengths as the input light source of the system. Multiple lasers of different wavelengths can simultaneously input lasers of one or more wavelengths, which are incident on the system along the same optical path. Furthermore, a dichroic mirror group 25 is provided in the image signal receiving unit to split the combined beam passing through the objective lens 14 into multiple beams, each of which enters a different channel. The multiple beams are adjusted by the lens group 26 (corresponding to the function of the lens 16), and then filtered out by the filter group 27 to obtain the spectral signals of the corresponding wavelengths. The multi-channel unit point detector 28 (corresponding to the function of the unit point detector 17) collects the spectral signals, generating optical data corresponding to the laser beams of different wavelengths. The dichroic mirrors, lenses, filters, and unit point detectors correspond to each other one-to-one.

[0085] Optionally, the combined light beam after heterodyne interference can be used to build a spatial light propagation path through optical elements, or can be coupled into an optical fiber to distribute light power to each channel through the optical fiber, and finally incident on the multi-channel unit point detector 28.

[0086] Alternatively, the objective lens 14 may be replaced by other types of lenses or other beam reduction optical elements. The dichroic mirrors in the dichroic mirror assembly 25 may be replaced by various optical elements such as semi-transparent and semi-reflective elements.

[0087] Figure 6 Schematic diagram of a microscopic imaging system according to an embodiment of the present invention, wherein the resolution exceeds the spot size limit and is continuously adjustable. Figure 6 As shown, a continuously variable zoom element 29 is used as a component of the divergence angle adjustment element. By continuously controlling the angle of the continuously variable zoom element 29, the divergence angle can be continuously adjusted, thereby achieving continuous adjustment of the system resolution. As the focal length of the continuously variable zoom element 29 changes, the relative position of the continuously variable zoom element 29 and the sample under test needs to be adaptively adjusted. A displacement control device can be added to move the continuously variable zoom element 29 as needed to ensure clear imaging.

[0088] Optionally, the continuous zoom element 29 is a zoom lens.

[0089] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A microscopic imaging system with a resolution exceeding the limit of spot size, characterized in that: The system comprises: a laser unit, for generating a laser beam; A target unit is provided in the optical path of the reference arm; the target unit includes a sample to be measured and a divergence angle adjustment element; the divergence angle adjustment element is used to adjust the divergence angle of the reference light source passing through the sample to be measured; A spatial radio frequency tag unit includes a reference arm optical path and a measurement arm optical path, which are used to perform optical heterodyne interference processing on the laser beam according to radio frequency drive signals of multiple frequencies to form a reference light source and a light spot array, respectively; after the reference light source is incident on the measured target unit, it is combined with the light spot array to generate a combined light beam; different light spots in the light spot array correspond to different light frequencies; an image signal receiving unit, configured to collect the combined light beam and generate light data; The data processing unit is used to receive the light data and perform image inversion processing on the light data to generate imaging data of the measured sample.

2. The system according to claim 1, wherein: The divergence angle adjustment element is a continuous zoom element.

3. The system according to claim 1, wherein: The spatial radio frequency tag unit further includes a radio frequency driver, a first spatial light modulator and a second spatial light modulator; the radio frequency driver is used to send radio frequency driving signals to the first spatial light modulator and the second spatial light modulator respectively; The first spatial light modulator is used to perform heterodyne interference processing on the laser beam according to the received RF drive signal to form a reference light source; the second spatial light modulator is used to perform heterodyne interference processing on the laser beam according to the received RF drive signal to form a light spot array.

4. The system according to claim 3, characterized in that The reference arm optical path also includes a beam shaping device and a first beam reduction element; the beam shaping device is used to shape the laser beam processed by the first spatial light modulator into a reference light source with uniform light intensity; the first beam reduction element is used to reduce the reference light source and then make it incident on the target unit to be measured.

5. The system according to claim 4, characterized in that The spatial radio frequency tag unit also includes a beam splitter and a beam combiner; The beam splitter is used to split the laser beam into a first laser beam and a second laser beam; the first laser beam is incident on the reference arm optical path; the second laser beam is incident on the measurement arm optical path; The beam combiner is used to combine the outgoing light beam after entering the target unit to be measured with the light spot array to generate a combined light beam.

6. The system according to any one of claims 1 to 5, characterized in that: The measured target unit further includes a displacement device; the measured sample is fixed on the displacement device; the displacement device is used to drive the measured sample to move to achieve push-scan imaging.

7. The system according to claim 6, characterized in that The spatial radio frequency tag unit further includes a second beam-contracting element; the image signal receiving unit includes a unit point detector; The second beam-contracting element is used to contract the combined light beam so as to be incident on the unit point detector; The unit point detector is used to perform photoelectric conversion on the incident light beam to generate light data.

8. The system according to claim 6, wherein: The data processing unit includes an acquisition card and an image inversion unit; the acquisition card is used to acquire the light data and perform analog-to-digital conversion to generate digital light data; the image inversion unit is used to convert the digital light data into the frequency domain to perform image inversion processing to generate imaging data of the sample under test.

9. The system according to claim 6, wherein: The laser unit includes a plurality of lasers; the plurality of lasers respectively generate laser beams of different wavelength bands; The image signal receiving unit includes a plurality of unit point detectors; the plurality of unit point detectors correspond one-to-one to the plurality of lasers and are used to respectively collect corresponding wavelength bands of the combined light beam to generate corresponding light data.

10. The system according to claim 9, characterized in that The image signal receiving unit further includes a plurality of filters; the plurality of filters correspond one-to-one to the plurality of unit point detectors, and are used to filter the combined light beams into corresponding wavelength bands and then incident on the corresponding unit point detectors.

Images