A microscopic imaging system that resolves beyond the spot size limit

Abstract

The present application relates to the technical field of microscopic imaging, and discloses a kind of microscopic imaging systems with resolution beyond spot size limit.The system includes: laser unit;Measured target unit is arranged in reference arm optical path;Measured target unit includes measured sample and divergence angle adjusting element;Divergence angle adjusting element is used to adjust the divergence angle size of reference light source passing through measured sample;Spatial radio frequency mark unit includes reference arm optical path and measurement arm optical path, for respectively carrying out optical heterodyne interference processing to laser beam according to radio frequency driving signal of multiple frequencies, respectively forming reference light source and spot array;Reference light source is combined with spot array after being incident measured target unit, generates combined beam;Image signal receiving unit is used to generate light data after combined beam is collected;Data processing unit is used to receive light data, and image inversion processing is carried out to light data, and the imaging data of measured sample is generated.The above scheme improves the resolution limit of system.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microscopic imaging technology, and in particular to a microscopic imaging system with resolution beyond the size limit of a laser spot. BACKGROUND

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

[0003] In related technologies, the smallest resolvable spot size that the actual optical system can achieve is the system resolution limit. Due to the non-ideal nature of optical system design and light source components, the actual spot size is often larger than the ideal spot size, resulting in poor system resolution. Furthermore, due to the limitations of processing technology and optical path, it is difficult to further reduce the laser spot to improve the resolution, and forcibly setting 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 system resolution and exceed the resolution of laser scanning imaging under the premise of a certain system spot size. SUMMARY

[0005] Therefore, the present application provides a microscopic imaging system with resolution beyond the size limit of a laser spot to solve the problem of limited microscopic imaging resolution. The technical solution is as follows:

[0006] A microscopic imaging system with resolution beyond the size limit of a laser spot is provided, which includes:

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

[0008] A measured target unit arranged in a 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;

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

[0010] An image signal receiving unit is configured to collect the post-beam combined light to generate light data;

[0011] A data processing unit is configured 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 adjusting element is a continuous zoom element.

[0013] In an optional embodiment, the spatial radio frequency marking unit further comprises a radio frequency driver, a first spatial light modulator, and a second spatial light modulator; the radio frequency driver is configured to send a radio frequency driving signal to the first spatial light modulator and the second spatial light modulator, respectively; the first spatial light modulator is configured to perform heterodyne interference processing on the laser beam according to the received radio frequency driving signal to form a reference light source; and the second spatial light modulator is configured to perform heterodyne interference processing on the laser beam according to the received radio frequency driving signal to form a spot array.

[0014] In an optional embodiment, the reference arm optical path further comprises a beam shaping device and a first beam shrinking element; the beam shaping device is configured to shape the laser beam processed by the first spatial light modulator into a reference light source with uniform light intensity; and the first beam shrinking element is configured to shrink the reference light source and then make the reference light source incident on the measured target unit.

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

[0016] The beam splitter is configured 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; and the second laser beam is incident on the measurement arm optical path.

[0017] The beam combiner is configured to combine the outgoing light beam after being incident on the measured target unit with the spot array to generate a beam combined light.

[0018] In an optional embodiment, the measured target unit further comprises a displacement device; the measured sample is fixed on the displacement device; and the displacement device is configured to move the measured sample to realize push-broom imaging.

[0019] In an optional embodiment, the spatial radio frequency marking unit further comprises a second beam shrinking element; and the image signal receiving unit comprises a unit point detector.

[0020] The second beam shrinking element is configured to shrink the beam combined light to make the beam combined light incident on the unit point detector; and the unit point detector is configured to perform photoelectric conversion processing on the incident light beam to generate light data.

[0021] In an optional implementation, the data processing unit comprises a collection card and an image inversion unit; the collection card is configured to collect the light data and perform analog-to-digital conversion to generate digital light data; and the image inversion unit is configured to convert the digital light data to a frequency domain to perform image inversion processing and generate the imaging data of the measured sample.

[0022] In an optional implementation, the laser unit comprises a plurality of lasers; and the plurality of lasers are configured to generate laser beams of different wave bands, respectively.

[0023] The image signal receiving unit comprises a plurality of unit point detectors; the plurality of unit point detectors correspond to the plurality of lasers one by one and are configured to collect corresponding wave bands of the combined beam to generate corresponding light data.

[0024] In an optional implementation, the image signal receiving unit further comprises a plurality of optical filters; the plurality of optical filters correspond to the plurality of unit point detectors one by one and are configured to filter the combined beam to the corresponding wave bands and then make the combined beam incident on the corresponding unit point detectors.

[0025] In an optional implementation, the system further comprises a host computer configured to visually display the imaging data.

[0026] The technical solution provided by the present application can include the following beneficial effects:

[0027] The microscopic imaging system provided by the present application breaks through the limitation of the resolution of the system by the size of the light spot. The microscopic imaging system comprises a laser unit, a measured target unit, a spatial radio frequency marking unit, an image signal receiving unit and a data processing unit. The laser unit is configured to generate a laser beam. The measured target unit is arranged in a reference arm optical path and comprises a measured sample and a divergence angle adjusting element configured to adjust the divergence angle of a reference light source passing through the measured sample. The spatial radio frequency marking unit comprises a reference arm optical path and a measurement arm optical path and is configured to perform optical heterodyne interference processing on the laser beam according to radio frequency driving signals of a plurality of frequencies to form a reference light source and a light spot array, respectively. The reference light source is combined with the light spot array after being incident on the measured target unit to generate a combined beam. Different light spots in the light spot array correspond to different light frequencies. The image signal receiving unit is configured to collect the combined beam to generate light data. The data processing unit is configured to receive the light data and perform image inversion processing on the light data to generate imaging data of the measured sample. By arranging the measured target unit in the reference arm optical path and arranging the divergence angle adjusting element to adjust the divergence angle of the reference light source passing through the measured sample, the resolution of the system can be adjusted, the limitation of the resolution of the system by the size of the light spot is broken, and the resolution limit of the system is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort based on these drawings.

[0029] Figure 1 is a structural schematic diagram of a resolution beyond the spot size limit microscopic imaging system according to an embodiment of the present application;

[0030] Figure 2 is a structural schematic diagram of another resolution beyond the spot size limit microscopic imaging system according to an embodiment of the present application;

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

[0032] Figure 4 is a structural schematic diagram of still another resolution beyond the spot size limit microscopic imaging system according to an embodiment of the present application;

[0033] Figure 5 is a structural schematic diagram of a multi-channel resolution beyond the spot size limit microscopic imaging system according to an embodiment of the present application;

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

[0035] Explanation of reference signs:

[0036] 1-laser unit; 2-first glass sheet; 3-second glass sheet; 4-third glass sheet; 5-fourth glass sheet; 6-fifth glass sheet; 7-beam splitter; 8-beam combiner; 9-first spatial light modulator; 10-second spatial light modulator; 11-first mirror; 12-second mirror; 13-beam expander; 14-objective lens; 15-samples to be measured; 16-lens; 17-unit point detector; 18-data processing unit; 19-upper computer; 20-radio frequency driver; 21-first convex lens; 22-second convex lens; 23-diaphragm; 24-beam homogenization mirror; 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 solutions of the present application will be described clearly and completely below in conjunction with the drawings. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0038] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation of the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0039] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

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

[0041] Figure 1 is a structural schematic diagram of a resolution super-spatial resolution microscopic imaging system according to an embodiment of the present application. As shown in the figure, in the resolution super-spatial resolution microscopic imaging system, a laser unit, a measured target unit, a spatial radio frequency marker unit, an image signal receiving unit and a data processing unit are included. Figure 1

[0042] The 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 adjusting element; the divergence angle adjusting element is used to adjust the divergence angle of the reference light source passing through the measured sample.

[0044] ​The spatial radio frequency marking unit comprises a reference arm optical path and a measurement arm optical path, and is used for respectively performing optical heterodyne interference processing on the laser beam according to radio frequency driving signals of multiple frequencies to respectively form a reference light source and a spot array; the reference light source is combined with the spot array after being incident on the measured target unit to generate a combined beam; and different spots in the spot array correspond to different light frequencies.

[0045] The image signal receiving unit is used for generating optical data after collecting the combined beam.

[0046] The data processing unit is used for receiving the optical data and performing image inversion processing on the optical data to generate imaging data of the measured sample.

[0047] Figure 1 The working principle of the illustrated microscopic imaging system which breaks through the spot size limit is as follows:

[0048] The laser unit is used for generating a laser beam as a light source of the system, and the laser beam is modulated or shaped after entering the optical path and then irradiated on the measured sample to generate an image signal. Optionally, the laser unit comprises a laser which has a narrow line width and high stability, and the formed spot shape can be circular, elliptical, rectangular and the like, and different shapes correspond to different initial resolutions.

[0049] The spatial radio frequency marking unit is used for implementing the spatial radio frequency marking technology which is a technology for marking spatial position coordinates by using frequency signals, and is implemented by using a heterodyne interference in the optical field, such as a Mach-Zehnder interference optical path. Specifically, the heterodyne interference optical path comprises a reference arm optical path and a measurement arm optical path, and the laser beam is divided into two paths and respectively enters the reference arm optical path and the measurement arm optical path. The reference arm optical path realizes shaping of the incident corresponding laser beam to obtain a reference light source which is incident on the measured target unit, and the incident light irradiates on the measured sample. The measured sample has different transmission rates for different spatial positions, so that the light intensity of the corresponding position is attenuated, thereby weakening the frequency domain signal amplitude of the corresponding position after subsequent frequency mixing and affecting the subsequently generated image of the measured sample; and the measurement arm optical path realizes spatial modulation of the incident corresponding laser beam, introduces frequency shift related to the driving frequency of the radio frequency driving signal for the laser spot of the laser beam, modulates the laser spot into a one-dimensional or two-dimensional spot array, and the spot interval and the number of the spot array are related to the radio frequency driving signal. Finally, the reference light source and the spot array of the measured target unit are combined to generate a frequency mixing signal, realize heterodyne interference of the one-dimensional or two-dimensional spot array, and each spot in the spot array has a specific frequency mixing envelope after the heterodyne interference, each frequency mixing envelope has a specific frequency, and the spatial position of the spot is marked by using the frequency mixing frequency.

[0050] Since the reference light source passing through the measured sample in the measured target unit is divergent light, a divergence angle adjusting element is arranged in the measured target unit to adjust the divergence angle of the reference light source passing through the measured sample. The divergence ability of the divergence angle adjusting element is a key to determine the system resolution. By adjusting the divergence angle of the divergence angle adjusting 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, so as to realize the microscopic imaging with resolution beyond the spot size limit.

[0051] The image signal receiving unit is used to collect the beat frequency light signal in the beam and convert the collected light signal into an electrical signal to obtain optical data, which is a time-domain voltage signal with optical information. The data processing unit receives the optical data output by the image signal receiving unit and processes the optical data by running an image inversion algorithm to realize image inversion and obtain imaging data of the measured sample.

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

[0053] Optionally, the divergence angle adjusting 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, and the system resolution can be adjusted.

[0054] In an optional embodiment, the divergence angle adjusting element is a continuous zoom element. By using the continuous zoom element as the divergence angle adjusting element, the divergence angle of the passing laser beam can be continuously adjusted, and the magnification of the microscopic image can be correspondingly and continuously adjusted, so as to continuously adjust the system resolution, improve the adjustment accuracy, and further improve the accuracy of the microscopic imaging.

[0055] In an optional embodiment, the spatial radio frequency marker 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 a radio frequency driving signal to the first spatial light modulator and the second spatial light modulator, respectively. The first spatial light modulator is configured to perform heterodyne interference processing on the laser beam according to the received radio frequency driving signal to form a reference light source. The second spatial light modulator is configured to perform heterodyne interference processing on the laser beam according to the received radio frequency driving signal to form a spot array.

[0056] The spatial light modulator is a device for modulating the spatial distribution of light waves. Under the control of an electrical driving signal, the spatial light modulator changes the frequency, amplitude or intensity, phase, polarization state, etc. of the spatial light distribution. The spatial light modulator includes an acousto-optic deflector AOD (Acousto-Optic Deflector), an acousto-optic frequency shifter AOFS (Acousto-Optic Frequency Shifters), an electro-optic modulator EOM (Electro-Optic Modulator), and an acousto-optic modulator AOM (Acousto-Optic Modulator), etc. The input of the spatial light modulator is a radio frequency driving signal. Different frequencies of the radio frequency driving signal will drive the spatial light modulator to produce different modulation effects. By applying different frequency components of the radio frequency driving signal to the spatial light modulator through the radio frequency driver, different parameters can be controlled. The spacing and number of the light spots of the modulated light spot array are related to the frequency component spacing and number of the radio frequency driving signal applied to the second spatial light modulator. A single frequency or a multi-frequency composite radio frequency driving signal can also be input. The multi-frequency composite radio frequency driving signal loaded on the spatial light modulator can be regarded as the superposition of the individual effects of each single frequency radio frequency driving signal contained in the multi-frequency composite radio frequency driving signal at the same time. The more the number of frequencies in the composite radio frequency driving signal, the worse the effect of each frequency.

[0057] For example, an acousto-optic deflector AOD is used as a spatial light modulator to apply a certain frequency shift to the incident light. The frequency shift size is the same as the frequency size of the radio frequency driving signal applied to the acousto-optic deflector AOD. The outgoing light has a certain deflection angle relative to the incident light. The deflection angle size is related to the frequency size of the radio frequency driving signal applied to the acousto-optic deflector AOD. When a single acousto-optic deflector AOD is used, a single frequency radio frequency driving signal can be sequentially applied to the acousto-optic deflector AOD to realize point scanning. Alternatively, a multi-frequency composite radio frequency driving signal can be simultaneously applied to the acousto-optic deflector AOD to generate a one-dimensional light spot array. When two acousto-optic deflectors AODs are used and placed orthogonally, a multi-frequency composite radio frequency driving signal can be simultaneously applied to the two acousto-optic deflectors AODs to generate a two-dimensional light spot array.

[0058] Optionally, the first spatial light modulator can also be replaced by a spatial light modulator group composed of multiple spatial light modulators, so that the outgoing 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 composed of multiple spatial light modulators, for example, a third spatial light modulator and a fourth spatial light modulator placed orthogonally to form a spatial light modulator group, so as to modulate the laser light spots of the incident second laser beam into a two-dimensional light spot array.

[0060] In an optional embodiment, the reference arm light path further comprises a beam shaping device and a first beam shrinking element; the beam shaping device is configured to shape the laser beam processed by the first spatial light modulator into a reference light source with uniform light intensity; and the first beam shrinking element is configured to shrink the reference light source and then make the reference light source incident on the measured target unit, so as to concentrate the light energy on the test area of the measured sample as much as possible, thereby fully utilizing the light energy. The reference light source is a two-dimensional surface light source. The shaped reference light source can cover the spot array, so as to ensure that each spot can realize heterodyne interference, and each spot in the spot array has a specific beat envelope after the heterodyne interference, each beat envelope has a specific frequency, and the beat frequency is used to mark the spatial position of the spot.

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

[0062] Optionally, the beam shaping device comprises a beam expander and a light barrier, the beam expander is configured to enlarge the laser spot of the laser beam processed by the first spatial light modulator, so as to use the enlarged laser spot with relatively concentrated and flat energy as the reference light source. The light barrier is configured to adjust the light flux of the laser beam passing through the beam expander, optimize the imaging quality, shape filtering, etc., and shape the incident laser beam together with the beam expander to obtain a reference light source with uniform light intensity. A Powell prism group, a cylindrical lens group, a commercial beam shaper, a beam homogenization mirror, etc. can also be used as the beam shaping device to shape the incident laser beam.

[0063] In an optional embodiment, the spatial radio frequency marking unit further comprises 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, the first laser beam is incident on the reference arm light path, and the second laser beam is incident on the measurement arm light path. The beam combiner is configured to combine the outgoing beam after the measured target unit with the spot array to generate a beat frequency, thereby generating a combined beam.

[0064] Optionally, a first glass sheet is arranged between the laser unit and the beam splitter, the first glass sheet is a 1 / 2 glass sheet, the laser unit generates laser beams with different polarization states, and the beam splitter is a polarization beam splitter. The laser beam emitted by the laser unit is first split into two beams of P light (Parallel-polarized light, corresponding to the first laser beam) and S light (Perpendicular-polarized light, corresponding to the second laser beam) with perpendicular polarization states after passing through the first glass sheet and the polarization beam splitter. By rotating the first glass sheet, the proportion of the first laser beam and the second laser beam to the laser beam can be changed, so as to adjust the light intensity of the first laser beam and the light intensity of the second laser beam.

[0065] In an alternative embodiment, the measured target unit further comprises 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 realize push-broom imaging.

[0066] Optionally, the displacement device further comprises a clamping structure for fixing the measured sample. The displacement device can drive the measured sample to move in multiple axes to realize high-precision and high-stability driving of the measured sample, and realize one-dimensional linear array push-broom and wobble-scan imaging when the light spot array is a one-dimensional array, and realize two-dimensional surface array push-broom and wobble-scan imaging when the light spot array is a two-dimensional array. The measured sample can move perpendicularly to the optical path between the light beam shaping device and the beam combiner to realize stitching imaging of a measured sample with a larger measured surface. Illustratively, the displacement device is an electric displacement stage.

[0067] Optionally, when the measured sample itself has single-axis or multi-axis movement capability, for example, the measured sample is a flow cytometer, the displacement device is not needed, and the light path of the flow cytometer is directly placed in the measured target area, and the flow axis is used as the displacement axis to realize push-broom imaging. When the laser spot of the measurement arm optical path of the heterodyne interference optical path is modulated into a two-dimensional light spot array, and the laser spot of the reference arm optical path is shaped into a surface light source, the displacement device can not be added, and video imaging of a dynamic scene can be directly realized.

[0068] In an alternative embodiment, the spatial radio frequency marking unit further comprises a second beam condensing element. The image signal receiving unit comprises a unit point detector. The second beam condensing element is configured to condense the combined beam to a size that can be incident on a target surface of the unit point detector, and to ensure that the focal points of the spot array of the measurement arm optical path and the measured sample pattern formed by the reference light source of the reference arm optical path after passing through the measured sample are on the unit point detector, so as to be incident on the unit point detector. The unit point detector is configured to receive the incident light beam to obtain a time domain optical signal, to perform photoelectric conversion processing on the time domain optical signal obtained based on the incident light beam, and to generate optical data converted into an electrical signal. The unit point detector is a non-imaging unit detector, which can only sense the intensity of the light signal, and is widely used in the fields of confocal, two-photon and single-molecule imaging. For example, the unit point detector is a photomultiplier tube (PMT), a photodiode (PD), an avalanche photodiode (APD) or a single photon avalanche diode (SPAD), and different unit point detectors can be selected as needed according to different measured samples. Compared with commonly used imaging detectors (such as charge coupled devices (CCD) and complementary metal oxide semiconductor (CMOS)), the unit point detector has the advantages of high detection sensitivity and high frame rate, and has obvious speed and sensitivity advantages in microscopic imaging applications at a small scale. The time domain signal of the unit point detector is an intensity signal of the optical beat frequency signal generated by the heterodyne interference optical path after passing through the measured sample, and is a superimposed signal of a plurality of frequencies and amplitudes, which represents the total power of the light signal incident on the target surface of the detector varying with time.

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

[0070] Optionally, the relative position between the imaging focal point position and the target surface of the unit point detector is adjusted by adjusting the distance between the divergence angle adjusting element and the measured sample, so as to ensure that the focal points of the light beams of the reference arm optical path and the measurement arm optical path are at the same target position after beam combination, and the target surface of the unit point detector is placed at the target position.

[0071] In an alternative embodiment, the data processing unit comprises a collection card and an image inversion unit. The optical data output by the image signal receiving unit is a time-domain voltage signal with optical information, which is an analog signal. The collection card is used to collect 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 collection card, convert the digital optical data to 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 value is equal to the signal strength to generate the 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 realizes intensity inversion of the optical signal at the corresponding spatial position by using the amplitude intensity of the specific frequency point of the frequency domain signal. The frequency domain assignment of a one-dimensional spot array is inverted to one-dimensional image data, and the frequency domain assignment of a two-dimensional spot array is inverted to two-dimensional image data. Alternatively, the time-domain signal is converted into a frequency-domain signal by Fourier transform.

[0072] Alternatively, the collection card is a high-speed data collection board card, and the sampling rate is at least greater than or equal to 2 times the maximum frequency of the beat envelope, and the bandwidth covers all envelope frequencies. The image inversion unit can be implemented on a high-speed hardware platform, such as a field programmable gate array (FPGA), a ZYNQ (a fully programmable system on a chip), a central processing unit (CPU), a graphics processing unit (GPU), and other high-speed data processing devices, to perform real-time and high-throughput image inversion. For example, the image inversion unit runs a Fourier transform algorithm on a high-speed hardware platform to convert the time-domain signal collected by the collection card from a unit point detector into a frequency-domain signal. Each beat envelope frequency corresponds to a frequency point, a specific spot, and a specific spatial position of a sample under test, i.e., a pixel coordinate in the image. The peak strength of the frequency represents the signal strength of the point, which represents the transmittance of the sample under test at that point. The frequency value and the peak strength at the frequency value are used to invert the image coordinates and pixel values to achieve imaging.

[0073] In an optional embodiment, the laser unit comprises a plurality of lasers; the plurality of lasers respectively generate laser beams of different wavebands. For different samples to be measured, one or more wavebands of lasers can be selected as needed to generate corresponding laser beams input into the optical path. Correspondingly, when the plurality of laser beams are simultaneously incident, the image signal receiving unit comprises a plurality of unit point detectors corresponding to the plurality of lasers, for respectively collecting corresponding wavebands of the combined beam to generate corresponding light data, so as to realize simultaneous collection of multi-spectral signals.

[0074] In an optional embodiment, the image signal receiving unit further comprises a plurality of optical filters corresponding to the plurality of unit point detectors, the optical filters only collect spectral signals of corresponding wavebands, so as to realize extraction of spectral signals of the sample to be measured, and the optical filters are used to respectively filter the combined beam to corresponding wavebands and then make the combined beam incident on the corresponding unit point detector.

[0075] In an optional embodiment, the system further comprises a host computer, which is used to visually display the imaging data and convert the imaging data into an image displayed on a screen. The host computer can also serve as a control terminal to realize functions such as control, task scheduling, image display, and instruction issuing between multiple devices of the system.

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

[0077] Figure 2 is a structural schematic diagram of a resolution beyond the spot size limit microscopic imaging system according to an embodiment of the present application. As shown in Figure 2 In the resolution beyond the spot size limit microscopic imaging system, a laser beam is generated by a laser unit 1, the laser beam is adjusted in polarization state by a first glass sheet 2 and then enters a beam splitter 7 to be split into a first laser beam and a second laser beam. The first laser beam changes the polarization state by a second glass sheet 3 and then enters a first spatial light modulator 9, the first spatial light modulator 9 modulates the corresponding laser beam incident according to a radio frequency driver 20 to send a radio frequency driving signal, and the first spatial light modulator emits the laser beam. The emitted laser beam of the first spatial light modulator is reflected by a first mirror 11 to a beam expander 13 for beam expansion, then enters an aperture 23 for shaping, and then enters a first convex lens 21 (corresponding to a first beam narrowing element) for beam narrowing and then enters a sample to be measured 15. The emitted beam of the sample to be measured 15 adjusts the divergence angle and the imaging focus position by a second convex lens 22 (corresponding to a divergence angle adjusting element), and then adjusts the polarization state by a fourth glass sheet 5 and then enters a beam combiner 8; the second laser beam adjusts the polarization state by a third glass sheet 4 and then is reflected by a second mirror 12 to a second spatial light modulator 10 Figure 2The laser beams emitted by the first spatial light modulator pass through the fifth glass sheet 6 to adjust the polarization state and then enter 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 shrinking element) in sequence to be shrunk and then enter the unit point detector 17 to generate optical data. The data processing unit 18 collects the optical data and performs image inversion processing to generate imaging data, which 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 radio frequency driver and the data processing unit respectively to coordinate the system operation.

[0078] The first glass sheet 2, the second glass sheet 3, the third glass sheet 4, the fourth glass sheet 5 and the fifth glass sheet 6 adjust the polarization state of the laser beams passing through them, so as to meet the polarization state requirements of the spatial light modulator and the interference light path, thereby ensuring the maximum interference efficiency. The positions and quantities of the glass sheets can be adjusted as needed.

[0079] The purpose of using the beam expander 13 to expand the incident light spot is to adjust the size of the laser spot to meet the size requirements of the light diaphragm 23 / beam homogenization mirror 24 for the incident light spot. The beam shaping module can shape the Gaussian spot into a flat-top light, thereby ensuring the high uniformity of the spot energy.

[0080] Optionally, the measured sample can be a fixed pattern, which is moved up and down or left and right by the displacement device to realize inversion imaging. The measured sample can also have a moving property, for example, when a flow cytometer is used for single cell analysis, the flow cytometer test pipeline can be fixed on the displacement device, and the displacement device is no longer used to drive the pipeline to move. Instead, the flow direction of the flow cytometer is used as the scanning axis, and combined with the two-dimensional array spot array, video imaging can be realized.

[0081] Optionally, the second convex lens 22 is used as a divergence angle adjusting element. Using second convex lenses 22 with different focal lengths can change the size of the unit size of the measured sample incident on the objective lens 14, and the number of line spots corresponding to different numbers of unit point detectors after beam shrinking. Figure 3 The imaging result schematic diagram according to the embodiment of the present application is shown in FIG. 2, and the imaging result schematic diagram when the convex lens with a focal length of 10 mm is used is shown in FIG. 3. Figure 3 As shown in FIG. 3, when the convex lens with a focal length of 10 mm is used, the image “H” with a width of 45 um on the measured sample corresponds to 5 light spots with a size of 12 um (1 / e^2) on the target surface of the unit point detector, thereby realizing a microscopic imaging scheme using a 12 um spot to achieve a 9 um imaging resolution.

[0082] The microscopic imaging system provided by the embodiment has resolution beyond the size limit of a light spot, and comprises a laser unit, a measured target unit, a spatial radio frequency marking unit, an image signal receiving unit and a data processing unit. The laser unit is used to generate a laser beam. The measured target unit is arranged in a reference arm light path and comprises a measured sample and a divergence angle adjusting element, which is used to adjust the divergence angle of the reference light source passing through the measured sample. The spatial radio frequency marking unit comprises a reference arm light path and a measurement arm light path, and is used to respectively perform optical heterodyne interference processing on the laser beam according to radio frequency driving signals of multiple frequencies, so as to respectively form a reference light source and a light spot array. The reference light source is combined with the light spot array after being incident on the measured target unit, so as to generate a combined beam. Different light spots in the light spot array correspond to different light frequencies. The image signal receiving unit is used to generate light data after the combined beam is collected. The data processing unit is used to receive the light data and perform image inversion processing on the light data, so as to generate imaging data of the measured sample. By arranging the measured target unit in the reference arm light path and arranging the divergence angle adjusting element to adjust the divergence angle of the reference light source passing through the measured sample, the resolution of the system can be adjusted, and the resolution limit of the system is broken, so that the resolution limit of the system is greatly improved.

[0083] Figure 4 Fig. 1 is a structural schematic diagram of a microscopic imaging system with resolution beyond the size limit of a light spot according to an embodiment of the present application. Figure 4 Compared with Figure 2 , the difference lies in that a beam homogenization mirror 24 is used to replace the diaphragm 23 to shape the incident laser beam.

[0084] Figure 5 Fig. 2 is a structural schematic diagram of a multi-channel microscopic imaging system with resolution beyond the size limit of a light spot according to an embodiment of the present application. Figure 5 In the laser unit 1 in Fig. 1, multiple lasers are contained, different waveband laser beams are generated by the multiple lasers as input light sources of the system, and the multiple different waveband lasers can input one or more waveband lasers at the same time, which are incident into the system along the same light path, and then the combined beam passing through the objective lens 14 is divided into multiple beams by a dichroic mirror group 25 arranged in the image signal receiving unit, each beam enters a different channel, the multiple beams are adjusted by a lens group 26 (corresponding to the function of the lens 16), and then the corresponding waveband spectrum signals are filtered out by a filter group 27, and the light data corresponding to the different waveband laser beams are generated by a multi-channel unit point detector 28 (corresponding to the function of the unit point detector 17). The dichroic mirror, the lens, the filter and the unit point detector are one-to-one corresponding.

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

[0086] Optionally, objective lens 14 can be replaced by other types of lenses or various other beam-constricting optical elements. The dichroic mirror in dichroic mirror group 25 can be replaced by various optical elements such as semi-transparent and semi-reflective mirrors.

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

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

[0089] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A microscopic imaging system with resolution exceeding the limitations of light spot size, characterized in that, The system includes: The laser unit is used to generate a laser beam; The spatial radio frequency marking 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 spot array respectively; The target unit under test is set in the optical path of the reference arm; the target unit under test includes a sample under test 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 under test; the divergence angle adjustment element is a continuous zoom element; after the reference light source is incident on the target unit under test, it is combined with the light spot array to generate a combined beam; different light spots in the light spot array correspond to different optical frequencies; An image signal receiving unit is used to acquire the combined beam and generate optical data; The data processing unit is used to receive the optical data and perform image inversion processing on the optical data to generate imaging data of the sample under test.

2. The system according to claim 1, characterized in that, The spatial radio frequency tagging 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 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 beam according to the received radio frequency drive signal to form a light spot array.

3. The system according to claim 2, characterized in that, The reference arm optical path further includes a beam shaping device and a first beam shrinking 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 shrinking element is used to shrink the reference light source before it is incident on the target unit under test.

4. The system according to claim 3, characterized in that, The space radio frequency marking 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 beam after it is incident on the target unit under test with the spot array to generate a combined beam.

5. The system according to any one of claims 1 to 4, characterized in that, The target unit under test further includes a displacement device; the sample under test is fixed on the displacement device; the displacement device is used to move the sample under test to achieve push-broom imaging.

6. The system according to claim 5, characterized in that, The spatial radio frequency marking unit further includes a second beam-shrinking element; the image signal receiving unit includes a unit point detector. The second beam-shrinking element is used to shrink the combined beam so that it is incident on the unit point detector; The unit point detector is used to perform photoelectric conversion processing on the incident light beam to generate optical data.

7. The system according to claim 5, characterized in that, The data processing unit includes a data acquisition card and an image inversion unit; the data 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 convert the digital optical data to the frequency domain for image inversion processing to generate imaging data of the sample under test.

8. The system according to claim 5, characterized in that, The laser unit includes multiple lasers; the multiple lasers generate laser beams of different wavelengths. The image signal receiving unit includes multiple unit point detectors; each of the multiple unit point detectors corresponds to one of the multiple lasers and is used to collect the corresponding wavelength band of the combined beam to generate corresponding optical data.

9. The system according to claim 8, characterized in that, The image signal receiving unit also includes multiple filters; each of the multiple filters corresponds to one of the multiple unit point detectors, and is used to filter the combined beam to the corresponding wavelength band before it is incident on the corresponding unit point detector.

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