A method for identifying macrophages in alveolar lavage fluid by terahertz near-field imaging

Abstract

The present application relates to biomedical micro-imaging and cell pathology detection technical field, especially to a kind of alveolar lavage fluid macrophage terahertz near-field imaging identification method.Its technical scheme includes the following steps: the unstained alveolar lavage fluid cell smear sample is fixed on the object table, the sample is observed under bright field using optical microscope, the suspected micro area with volume difference or nuclear shape shadow characteristics is locked, and the near-field probe is focused to the suspected micro area;The suspected micro area is scanned in a large range using a terahertz near-field imaging system, and surface topography images and terahertz internal tomography images are obtained.According to the size characteristics of the target object in the image and the uniformity characteristics of the terahertz signal, the background interference cells are excluded.The present application provides a terahertz near-field imaging method that can quickly and objectively identify different physiological state macrophages without staining, based on the dual-mode signals of topography elevation and optical voltage.

Description

Technical Field

[0001] This invention relates to the field of biomedical microscopic imaging and cell pathology detection technology, and in particular to a terahertz near-field imaging identification method for macrophages in bronchoalveolar lavage fluid. Background Technology

[0002] Cytological analysis of bronchoalveolar lavage fluid (BALF) is a core tool for the clinical diagnosis of pulmonary inflammation, interstitial lung disease, and microenvironmental infections. As the core unit for immune defense in the alveolar region, the atypia of macrophages and their phagocytic degradation state directly reflect the evolutionary characteristics of the pulmonary pathological environment. Therefore, accurately identifying macrophages in bronchoalveolar lavage fluid smears and effectively assessing their morphology, activation state, and phagocytic function is of significant guiding importance for the clinical diagnosis of respiratory diseases. However, traditional cytological diagnostic methods suffer from insurmountable technical bottlenecks.

[0003] First, in order to identify macrophages in the complex background of lavage fluid, multiple complex chemical staining procedures (such as Wright-Giemsa staining) must be performed on cell smears. This pretreatment usually takes more than an hour, which not only greatly reduces the efficiency of clinical testing, but also the irreversible intervention of organic dyes can completely destroy the intrinsic bioelectromagnetic state of the cells, making the cells unusable for subsequent non-destructive testing or spectroscopic analysis.

[0004] Secondly, even after staining, traditional optical microscopes are limited by the wavelength of visible light, with an insurmountable micrometer-level diffraction limit, making it absolutely impossible to resolve nanoscale cell membrane pseudopodia protrusions. Although terahertz waves have unique advantages of being non-destructive, label-free, and highly sensitive, traditional far-field terahertz imaging technology is limited by the diffraction limit, with a spatial resolution of only millimeters to tens of micrometers, which is insufficient to meet the high-resolution detection requirements at the single-cell scale.

[0005] Furthermore, due to the lack of penetrability of photons, traditional two-dimensional planar imaging cannot capture the intrinsic physicochemical information of subcellular organelles such as phagocytic inclusion bodies and dense regions within the nucleus. When faced with unstained, original, translucent smears, ordinary bright-field optical microscopes can barely detect the macroscopic edges of some large cells and the faint shadows of kidney-shaped nuclei, but due to the lack of contrast and insufficient depth of field, they cannot provide quantitative and reproducible physical evidence for the complex phagocytic vacuolation characteristics within cells, which can easily lead to subjective misjudgment.

[0006] In recent years, terahertz scattering scanning near-field optical microscopy based on atomic force microscopy has provided a new technical approach to overcome the aforementioned bottlenecks. This technique focuses terahertz waves onto the tip of a nanoscale metal probe, utilizing the electromagnetic interaction between the probe and the sample in the near-field range to simultaneously acquire nanoscale morphological elevation information of the sample surface and terahertz near-field scattering signals. This overcomes the optical diffraction limit, achieving nanoscale imaging and spectroscopy. Existing research has applied terahertz near-field imaging technology to fields such as single-bacterial identification, thyroid tumor tissue region identification, and cell imaging.

[0007] However, applying terahertz near-field imaging technology to the identification of macrophages in bronchoalveolar lavage fluid still faces the following technical challenges that urgently need to be addressed:

[0008] First, the cellular composition of bronchoalveolar lavage fluid is complex, containing not only target macrophages but also various background interfering cells such as lymphocytes and neutrophils. The primary challenge in establishing an efficient identification method is how to quickly and accurately locate suspected macrophages within this complex cell population.

[0009] Secondly, macrophages exhibit significant physiological heterogeneity—their volume, morphology, and intracellular structure change drastically from their normal resting state to their highly activated state. Establishing a set of criteria that can broadly recognize macrophages under different physiological states is a crucial problem that urgently needs to be solved.

[0010] Third, terahertz near-field imaging systems can output multiple physical signal channels, such as topographic elevation channels and optical voltage channels. However, how to effectively map these multidimensional physical parameters to the pathological characteristics of macrophages and establish a set of specific targeting criteria that can be stably output by the system software without relying on the absolute dielectric constant value is a technical challenge currently faced in the medical-engineering interdisciplinary field.

[0011] Fourth, existing terahertz near-field imaging methods are mostly aimed at microorganisms with relatively simple structures, such as bacteria, or at macroscopic samples such as tissue sections. They lack systematic identification schemes for single-cell levels, especially for eukaryotic cells with complex subcellular structures, such as macrophages.

[0012] In summary, how to utilize the nanoscale spatial resolution and sensitivity to the complex electromagnetic responses of biomolecules in terahertz near-field imaging systems to establish a high-throughput, non-destructive, and high signal-to-noise ratio targeted identification method for macrophages in bronchoalveolar lavage fluid is a pressing technical problem to be solved in this field. Therefore, this application proposes a terahertz near-field imaging identification method for macrophages in bronchoalveolar lavage fluid. Summary of the Invention

[0013] The purpose of this invention is to address the technical problems in macrophage identification and diagnosis in the prior art by proposing a terahertz near-field imaging method for macrophage identification in bronchoalveolar lavage fluid.

[0014] The technical solution of the present invention: a terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid, comprising the following steps:

[0015] Step 1: Fix the unstained bronchoalveolar lavage fluid cell smear sample onto the stage, observe the sample under bright field using an optical microscope, identify suspected micro-regions with volume differences or nucleus shadow characteristics, and drive the near-field probe to focus on the suspected micro-regions.

[0016] Step 2: Use a terahertz near-field imaging system to perform a large-scale scan of the suspected micro-region to obtain surface morphology images and terahertz internal tomographic images. Based on the size characteristics of the target object and the uniformity characteristics of the terahertz signal in the images, background interference cells are eliminated, and the spatial coordinates of the suspected macrophages are determined.

[0017] Step 3: Based on the spatial coordinates, reduce the scanning range to perform high-resolution scanning, and obtain high-resolution surface morphology elevation data and terahertz near-field scattering optical voltage signal of the target cells;

[0018] Step 4: Perform multimodal fusion analysis on the surface topography elevation data and the terahertz near-field scattering optical voltage signal. If the target cell simultaneously meets the following three characteristics, it is identified as a macrophage:

[0019] Feature 1: The three-dimensional morphology shows that its volume is larger than that of the background cells, and the cell membrane edge has irregular multi-pseudopodia-like protrusions;

[0020] Feature 2: In optical voltage images, there are localized low-level dark areas that are eccentrically distributed inside the cells;

[0021] Feature 3: In optical voltage images, there are dense micro-regions of voltage signal abrupt changes with dramatic light and dark contrast in the cytoplasm.

[0022] Optionally, the terahertz near-field imaging system is a scattering near-field scanning terahertz microscope, the operating frequency of which is 90-100 GHz; the probe is a metal probe that oscillates vertically in tapping mode, and its tip curvature radius is no greater than 20 nm.

[0023] Optionally, in step one, the coated sample is supported on a low-resistivity silicon wafer substrate, and a micro magnetic sheet is provided on the back of the low-resistivity silicon wafer, which is fixed to the stage by magnetic adsorption.

[0024] Optionally, in step two, the scanning window for the large-area scan is 40 μm × 40 μm to 60 μm × 60 μm, the scanning resolution is 512 × 512 pixels, and the scanning frequency is 0.4 Hz.

[0025] Optionally, in step two, the background interfering cells include lymphocytes and neutrophils. The exclusion criteria are: lymphocytes with a diameter of 7-10 μm and neutrophils with a diameter of 10-15 μm, and their signals are uniform in the terahertz internal tomographic image; while the physical major diameter of suspected macrophages is greater than 15 μm and the terahertz signal is mottled and unevenly distributed.

[0026] Optionally, in step three, the scanning window of the high-resolution scan is 23.5 μm × 23.5 μm to 45 μm × 45 μm, the scanning resolution is at least 512 × 512 pixels, and the scanning frequency is 0.2 Hz.

[0027] Optionally, in step four, in feature 1, the volume of the target cell corresponds to a major axis of 15 μm to 50 μm, and the cell surface has nanoscale elevation roughness.

[0028] Optionally, in step four, the local low-level dark area of ​​feature 2 characterizes the characteristic nucleus of macrophage, which includes the kidney-shaped nucleus and horseshoe-shaped nucleus in the resting state, as well as the compressed oval nucleus that is displaced to the cell edge by phagocytic vesicles in the activated state.

[0029] Optionally, in step four, the voltage signal mutation micro-region of feature 3 represents the phagocytic vacuoles formed in the cytoplasm of macrophages due to the phagocytosis of lipids, dust or cell debris, and the micro-regions appear as granular or honeycomb-like distributions in the image.

[0030] Optionally, the method can be used for in vitro identification of macrophages in bronchoalveolar lavage fluid for non-diagnostic purposes.

[0031] Compared with the prior art, this application includes at least one of the following beneficial technical effects:

[0032] The identification architecture established in this invention deeply explores the nanoscale physical feedback potential of near-field optical systems in the 100 GHz frequency band. It can not only depict the nanoscale pseudopodia roughness of cell surfaces through mechanical feedback, but also directly map the voltage contrast at different tomographic depths within the cell using the penetrability of terahertz waves. This fundamentally breaks through the limitations of diffraction and opens up a new quantitative approach for observing the physicochemical distribution inside living / normal single cells.

[0033] This invention, through ingenious transformation of parameter indicators, completely eliminates the awkwardness of equipment operation in experiments where it is difficult to accurately derive the absolute dielectric constant value. The solution transforms the medically determined standards of "phagocytic vacuolation" and "heterogeneous nucleus" into "voltage gradient mutation" and "low-level local dark contrast," which can be directly and stably output by software. This determination strategy has extremely strong broad-spectrum inclusiveness for both macrophages in the normal resting state and those in a highly activated state at the peak of phagocytosis.

[0034] Macrophage imaging primarily utilizes the nanometer-level high precision of an atomic force microscope's terahertz near-field imaging system. It employs a two-stage scanning strategy, first measuring a large area and then a small area, ensuring both accurate target cell localization and high-resolution surface and internal tomographic imaging. This imaging system achieves up to a thousandfold magnification, allowing for direct observation of the sample cell surface and different depth layers, overcoming the difficulties in examining subcellular structures caused by the low magnification and diffraction limit of traditional optical microscopes.

[0035] Traditional cytological identification methods require complex staining of samples. The imaging system in this invention can directly detect unstained sample smears with high imaging precision, clearly observing the irregular nuclear shape and phagocytic vacuoles characteristic of macrophages. The entire process, from slide mounting to imaging completion, takes less than one hour, significantly reducing sample preparation and staining steps, saving considerable time, and improving the overall efficiency of cytopathological identification.

[0036] This invention uses terahertz waves in the 100 GHz band for excitation. The non-ionizing and non-destructive irradiation of biological samples in this band ensures the non-destructive nature of the detection process, allowing the sample to still be subjected to other subsequent detections after imaging, and has good compatibility and repeatability.

[0037] In summary, this invention provides a terahertz near-field imaging method that requires no staining, is based on dual-modal signals of morphological elevation and optical voltage, and can rapidly and objectively identify macrophages in different physiological states. Attached Figure Description

[0038] Figure 1 This is a reference image of bronchoalveolar lavage fluid cells taken under a conventional optical microscope without staining in a bright field. In the image, the macroscopic boundaries of the cells and the shadow of the standard kidney-shaped nucleus are faintly visible in the upper left, but the fine pseudopodia and intracellular phagocytic structures cannot be distinguished.

[0039] Figure 2 This is a large-scale image of the physical morphology of the cell surface of macrophages within a 40 μm range;

[0040] Figure 3 It corresponds Figure 2 Preliminary screening image of intracellular tomography in the field of view;

[0041] Figure 4 This is a nanoscale elevation morphology of the cell surface of monocytes and macrophages in the normal resting state within the range of 23.5 μm;

[0042] Figure 5 This is a high-precision internal terahertz tomographic image of a monocyte / macrophage in its normal resting state within the 23.5 μm range;

[0043] Figure 6 This is a high-precision internal terahertz tomographic image of a highly activated macrophage at a field of view of approximately 45 μm. Detailed Implementation

[0044] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0045] Example

[0046] This embodiment discloses a terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid. The scattering near-field imaging system upon which this invention relies mainly consists of a 100 GHz terahertz excitation source, a focusing lens group, a metal probe with a curvature radius ≤20 nm, a detection demodulation module, and a back-end imaging terminal such as CSPM system software.

[0047] Step 1: Cross-scale linkage focusing and positioning. Obtain a completely unstained bronchoalveolar lavage fluid smear sample prepared by the hospital. The sample is supported on the surface of a low-resistivity silicon wafer and adsorbed onto a three-dimensional movable stage by a micro-magnetic plate on the back.

[0048] Activate the system's built-in optical microscope to perform macroscopic preliminary screening under bright field conditions, such as... Figure 1 As shown. Due to the high transparency of the cells, the operator uses a large, shadowy micro-area with a faintly visible "standard kidney-shaped" outline in the field of view to perform coarse screening and positioning. This micro-area is then moved vertically below the metal probe, the laser is turned on, and the probe's automatic frequency adjustment is activated, allowing the cantilever to smoothly advance the needle in tapping mode, reaching the near-field electromagnetic strong coupling region.

[0049] Step 2: Dual-channel large-area scanning initial screening. Fine-tune the optical path to focus the 100 GHz terahertz wave onto the needle tip, and perform the first large-area initial screening. Set the scanning window to 40 μm × 40 μm, the scanning frequency to 0.4 Hz, and the data resolution to 512 × 512 pixels.

[0050] System output after scanning Figure 2 and Figure 3 Based on the accuracy formula R=W / C, the imaging accuracy at this stage is 78.13 nm. By comparison, the diameters of lymphocytes and neutrophils are usually between 7-15 μm. If a target with a physical major axis exceeding 15 μm and exhibiting mottled electromagnetic reflection is identified, the coordinates of suspected macrophages can be accurately determined.

[0051] Step 3: Multi-state small-range precision measurement. For the coordinates locked in Step 2, perform a small-range precision scan:

[0052] Status A, standard monocytes / macrophages: The scan window was reduced to 23.5 μm × 23.5 μm, while maintaining a resolution of 512 × 512, and the frequency was reduced to 0.2 Hz. Output Figure 4 and Figure 5 The computational imaging accuracy reaches 45.9 nm, achieving extremely high resolution at the subcellular level.

[0053] State B, highly activated macrophages: When the cell volume is extremely expanded and fills the field of view, the scanning area is adjusted to approximately 45 μm × 45 μm, the measured size is 45118 nm × 45118 nm, the resolution is 512, and the output... Figure 6 The high-contrast internal image shown has an imaging resolution of approximately 87.9 nm, which is sufficient to resolve dense cavitation interfaces.

[0054] Step 4: Targeted Determination of Multimodal Specific Indicators. The system performs multimodal mapping between the AFM three-dimensional physical elevation parameters and the 110 GHz scattered signal voltage contrast parameters to perform targeted determination:

[0055] (1) Extraction of topographic elevation parameters: combined with Figure 4 Resting macrophages have a long diameter of 15 μm-30 μm, while activated monomers can expand to over 40 μm. Three-dimensional data revealed that their membrane edges exhibit irregular amoeboid pseudopodia extensions, and their surfaces are densely covered with nanoscale elevation roughness, significantly different from the smooth surface of ordinary leukocytes.

[0056] (2) Contrast extraction in dense nuclear regions: Terahertz waves are extremely sensitive to the refractive index of substances with highly aggregated nucleic acids. Figure 5 and Figure 6 In the middle, the core region produces specific absorption modulation of electromagnetic waves, which is consistently presented as a "dark contrast area" with a local low level in the voltage image. Figure 5 Clearly identified as eccentrically distributed kidney-shaped or horseshoe-shaped nuclei; Figure 6 In this study, the dark area was compressed to the lower left corner of the cell's outermost edge by a large number of endocytic foreign bodies, exhibiting an irregular, compressed state. This protocol broadly encompasses a variety of typical nuclei.

[0057] (3) Extraction of vacuolar cluster signal mutations: The complex permittivity of the lipids and dust encapsulated in the phagocytic vesicles is very different from that of the cytoplasm, which will cause strong mutations in the near field voltage. Figure 5 Scattered voltage gradient spots have already appeared in the mesocytoplasm; while at the peak of phagocytosis... Figure 6 Within the macrophage, the cytoplasm is filled with extremely dense, honeycomb-like microregions of high contrast. These microregions appear as bright spots with dramatic voltage-induced brightness variations. This dramatic optical voltage contrast, caused by differences in intrinsic physicochemical composition, constitutes the unique "dielectric fingerprint" of macrophages.

[0058] By combining the pseudopodia elevation parameters mentioned above, as well as the voltage mutation signals of the eccentric dense dark nucleus region and high-density vacuolar clusters, the target cells can be accurately identified as macrophages in a completely stain-free state, completely avoiding the limitations of subjective visual description.

[0059] Application Example 1

[0060] The above method was used to identify macrophages in bronchoalveolar lavage fluid samples from a clinically sourced patient. The sample was obtained from the bronchoalveolar lavage fluid of a patient suspected of having interstitial pneumonia. After fixing the unstained cell smear sample onto the stage, steps 1 to 4 were performed sequentially. The entire identification process, from sample loading to outputting the judgment result, took approximately 50 minutes. The imaging results clearly showed the amoeboid pseudopodia surface morphology of the target cells, the eccentrically distributed kidney-shaped dark areas, and the dense phagocytic vacuolar voltage mutation signals within the cytoplasm. Based on these three criteria, macrophages were accurately identified. Compared with traditional Wright-Giemsa staining followed by optical microscopy, this method eliminates the need for pre-staining treatment, improves imaging resolution by more than two orders of magnitude, and simultaneously acquires dual-modal physical information on both the nanoscale morphology of the cell surface and the distribution of intracellular subcellular organelles.

[0061] Application Example 2

[0062] The above method was used to identify highly activated macrophages. The sample was obtained from bronchoalveolar lavage fluid of a patient with a lung infection. In step 3, because the target cells were found to be significantly swollen in volume during the initial screening, the scanning window was switched to 45 μm × 45 μm for precise measurement. The imaging results showed that the cytoplasm was filled with honeycomb-like micro-regions with intense voltage contrast, while the dense nucleus was squeezed to one corner of the cell. Based on the three criteria, the cells were accurately identified as highly activated macrophages in the peak of phagocytic activity. This embodiment verifies the broad-spectrum identification capability of the method of the present invention for macrophages in different physiological states.

[0063] It is worth noting that this invention eliminates the need for any staining or labeling of bronchoalveolar lavage fluid cell smears, directly utilizing a terahertz near-field imaging system to detect unstained samples. This label-free method avoids altering the intrinsic dielectric properties and morphological structure of cells with chemical staining agents, resulting in detection results that more closely reflect the true physiological state of cells. Simultaneously, eliminating the pre-staining treatment significantly shortens the overall detection cycle, reduces operational complexity, and lowers reagent costs. Because terahertz waves are non-ionizing, the detection process is non-destructive to the sample, and the imaged cell smears can still be used for other subsequent analyses or verifications.

[0064] Furthermore, this invention establishes a dual-modal physical parameter fusion judgment system by simultaneously acquiring surface morphology elevation signals from the atomic force microscope channel and optical voltage signals from the terahertz near-field scattering channel. The surface morphology channel reflects the degree of pseudopodia extension and surface roughness at the cell membrane edge, while the optical voltage channel characterizes the differences in electromagnetic absorption in the nucleus region and the voltage contrast changes in phagocytic vesicles caused by abrupt changes in dielectric constant. These three physical characteristics are intrinsic properties unique to macrophages and can be directly quantified by the system, providing objective and clear judgment criteria and avoiding the uncertainty caused by relying on subjective visual experience for cell type identification under traditional bright-field optical microscopy.

[0065] Furthermore, this invention employs a two-stage scanning strategy: a large-scale initial screening followed by a small-scale precision measurement. The large-scale scanning can quickly eliminate interfering cells such as lymphocytes and neutrophils in complex backgrounds containing various white blood cells, based on size and terahertz signal uniformity, thus determining the spatial coordinates of suspected macrophages. The small-scale precision measurement then performs high-resolution imaging on the identified target to ensure clear resolution of subcellular structures. This strategy balances localization efficiency and imaging quality, adapting to macrophages of different sizes and activation levels, and effectively identifying both conventional resting and highly activated samples using a unified criterion.

[0066] The above specific embodiments are merely several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant teachings of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid, characterized in that, Includes the following steps: Step 1: Fix the unstained bronchoalveolar lavage fluid cell smear sample onto the stage, observe the sample under bright field using an optical microscope, identify suspected micro-regions with volume differences or nucleus shadow characteristics, and drive the near-field probe to focus on the suspected micro-regions. Step 2: Use a terahertz near-field imaging system to perform a large-scale scan of the suspected micro-region to obtain surface morphology images and terahertz internal tomographic images. Based on the size characteristics of the target object and the uniformity characteristics of the terahertz signal in the images, background interference cells are eliminated, and the spatial coordinates of the suspected macrophages are determined. Step 3: Based on the spatial coordinates, reduce the scanning range to perform high-resolution scanning, and obtain high-resolution surface morphology elevation data and terahertz near-field scattering optical voltage signal of the target cells; Step 4: Perform multimodal fusion analysis on the surface topography elevation data and the terahertz near-field scattering optical voltage signal. If the target cell simultaneously meets the following three characteristics, it is identified as a macrophage: Feature 1: The three-dimensional morphology shows that its volume is larger than that of the background cells, and the cell membrane edge has irregular multi-pseudopodia-like protrusions; Feature 2: In optical voltage images, there are localized low-level dark areas that are eccentrically distributed inside the cells; Feature 3: In optical voltage images, there are dense micro-regions of voltage signal abrupt changes with dramatic light and dark contrast in the cytoplasm.

2. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, The terahertz near-field imaging system is a scattering near-field scanning terahertz microscope, and its terahertz excitation source operates at a frequency of 90-100 GHz; the probe is a metal probe that oscillates vertically in tapping mode, and its tip curvature radius is no greater than 20 nm.

3. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, In step one, the coated sample is supported on a low-resistivity silicon wafer substrate. The back of the low-resistivity silicon wafer is provided with a micro magnetic sheet, which is magnetically attracted and fixed to the stage.

4. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, In step two, the scanning window for the large-area scan is 40 μm × 40 μm to 60 μm × 60 μm, the scanning resolution is 512 × 512 pixels, and the scanning frequency is 0.4 Hz.

5. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, In step two, the background interference cells include lymphocytes and neutrophils. The exclusion criteria are: lymphocytes with a diameter of 7-10 μm and neutrophils with a diameter of 10-15 μm, and their signals are uniform in the terahertz internal tomographic image; while the physical major diameter of suspected macrophages is greater than 15 μm and the terahertz signal is mottled and unevenly distributed.

6. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, In step three, the scanning window for the high-resolution scan is 23.5 μm × 23.5 μm to 45 μm × 45 μm, the scanning resolution is at least 512 × 512 pixels, and the scanning frequency is 0.2 Hz.

7. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, In step four, in feature 1, the volume of the target cell corresponds to a major axis of 15 μm to 50 μm, and the cell surface has nanoscale elevation roughness.

8. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, In step four, the local low-level dark area of ​​feature 2 represents the characteristic nucleus of macrophage, which includes the kidney-shaped nucleus and horseshoe-shaped nucleus in the resting state, as well as the compressed oval nucleus that is displaced to the cell edge by phagocytic vesicles in the activated state.

9. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, In step four, the voltage signal mutation micro-region of feature 3 represents the phagocytic vacuoles formed in the cytoplasm of macrophages due to the phagocytosis of lipids, dust or cell debris, and the micro-regions appear as granular or honeycomb-like distributions in the image.

10. The terahertz near-field imaging method for identifying macrophages in bronchoalveolar lavage fluid according to claim 1, characterized in that, The method is used for in vitro identification of macrophages in bronchoalveolar lavage fluid for non-diagnostic purposes.

Images