Marked light-assisted microscopic imaging method and device

Abstract

In the marking light-assisted microscopic imaging method and device, a marking light assembly emits marking light which passes through a spectroscope, and the marking light is focused on a target object to form a marking pattern; the mark pattern enters the image sensor through the lens assembly and the spectroscope, and a mark image of the target object is obtained on the image sensor; the microscopic imaging unit irradiates a target object through an imaging light source, and a microscopic image of the target object is obtained on an image sensor; adjusting the distance between the target object and the lens assembly, and judging whether the microscopic imaging assembly correctly focuses on the target object or not by judging whether the mark image is imaged on the image sensor or not; or the distance between the image sensor and the marking light assembly and the lens assembly is adjusted, and whether the microscopic imaging assembly focuses on the target object correctly or not is judged. In the application, the inventor proposes that the mark pattern is formed at the focus point by using the mark light, and the mark image is obtained by carrying out microscopic imaging on the mark pattern, so as to assist the traditional microscopic equipment in focusing or measuring the distance between different positions.

Description

Technical Field

[0001] This application belongs to the field of microscopic imaging technology, and particularly relates to a method and device for marker light-assisted microscopic imaging. Background Art

[0002] The applicant has proposed a series of Chinese patents, such as

[0003] 1. CN2020112669290, "Cell Analysis Method and System and Quantitative Method and System";

[0004] 2. CN2020112669182, "Imaging Method and System for Cell Suspension Samples and Kit";

[0005] 3. CN2020112669182, "Imaging Method and System for Cell Suspension Samples and Kit";

[0006] 4. CN2022104799126, "Fast Focusing Method for Microscopic Image Acquisition Device and Microscopic Image Acquisition Method";

[0007] 5. CN2023100423151, "Blood Imaging Analysis System and Method";

[0008] A brand-new technical solution is used to measure the content of target substances in blood, urine, and feces, opening up a brand-new technical route for detecting the formed components in suspension.

[0009] However, for the quantitative detection of the content of formed components in suspension based on a microscope, it not only requires accurately obtaining the images of the detected formed components, identifying and distinguishing the detection target substances, but also accurately calculating the volume of the liquid in which the detected target substances are located.

[0010] To accurately calculate the volume of the liquid in which the detected target substances are located, the accurate dimension of the height of the liquid is one of the key elements. Under the high-power microscope field of view, generally the corresponding area is less than 1 mm * 1 mm, the height of the liquid is between 0.1 mm and 0.5 mm, and the height error of the liquid is 1 micron. For a liquid height of 0.1 mm, the error has reached 1%. Therefore, it is necessary to improve the measurement accuracy of the liquid height as much as possible.

[0011] Generally, the suspension to be detected is placed in a detection cavity. Therefore, how to measure the height of the liquid is transformed into measuring the height of the detection cavity.

[0012] When the factory produces detection chips, the consistency and accuracy of the height of the detection cavity are controlled through the process. The accurate size of the detection cavity can also be obtained through accurate measurement. However, during the transportation, storage, and use of the detection chips, the size will change due to environmental temperature or external pressure. If this change reaches more than 1 micron, the chips will basically lose their use value.

[0013] In a common wide-field optical microscope, the entire specimen is illuminated by the light of a mercury arc lamp or a xenon lamp, and the image can be directly observed with the naked eye. At the same time, the fluorescence from other regions outside the focus interferes greatly with the structure, especially when the thickness of the specimen is more than 2um, the influence is more obvious.

[0014] Such as Figure 11 , 12 , is the schematic diagram of a laser confocal microscope. The confocal laser scanning microscope (CLSM) consists of two parts: a confocal microscope and a femtosecond infrared laser Verdi / Mira. It is the combination of an optical microscope and modern laser technology, high-sensitivity detection technology, scanning control technology, microcomputer image processing technology, fluorescence and labeling technology. CLSM has opened up a new way to observe the structure of living cells and the biological changes of specific molecules and ions in the life sciences, and has become a new generation of powerful research tools in the fields of molecular cell biology, neuroscience, pharmacology, genetics, etc.

[0015] The laser confocal microscope breaks away from the field light source and local plane imaging mode of the traditional optical microscope. It uses a laser beam as the light source. The laser beam passes through the illumination pinhole, is reflected by the beam splitter to the objective lens, and is focused on the sample. Each point on the focal plane of the specimen is scanned. If there is a fluorescent substance that can be excited in the tissue sample, the fluorescence emitted after excitation directly returns to the beam splitter along the original incident light path. When passing through the detection pinhole, it is first focused, and the focused light is detected and collected by a photomultiplier tube (PMT), and the signal is transmitted to the computer and displayed as an image on the computer monitor after processing. In this optical path, only the light on the focal plane can pass through the detection pinhole, and the light from the regions outside the focal plane is defocused on the detection pinhole plane and cannot pass through the pinhole. Therefore, the background of the non-observation point is black, the contrast increases, and the imaging is clear. Since the illumination pinhole and the detection pinhole are conjugate with respect to the focal plane of the objective lens, the points on the focal plane are simultaneously focused on the illumination pinhole and the detection pinhole, and the points outside the focal plane will not be imaged at the detection pinhole, that is, confocal. Scanning the sample with a laser as the light source and focusing twice during this process, it is called a laser scanning confocal microscope.

[0016] The laser confocal microscope has a high cost and a complex usage process. It does not have the field light source of the traditional optical microscope and cannot take plane images.

[0017] Figure 13It is a schematic diagram of the principle of Chinese Patent Application No. 202110127353.8, with the application title "A Laser Ranging Method, a Focusing Method, a Laser Ranging System and a Focusing System". In the microscope imaging channel, a laser ranging device 9 is added, and the distance is measured by measuring the time difference between the emitted laser and the received laser. However, this method is difficult to measure the height of a liquid and also difficult to measure the height of a detection cavity. Summary of the Invention

[0018] In this application, the inventor proposes to form a marking pattern at the focal point with a marking light, and obtain a marking image by performing microscopic imaging on the marking pattern to assist the traditional microscopic device in focusing. Further, by focusing at different positions, the distance measurement between different positions is completed.

[0019] A microscopic imaging device with marking light assistance includes a microscopic imaging unit and a marking light unit; the microscopic imaging unit includes an image sensor, a lens assembly, an imaging light source, and a Z-axis adjustment assembly; the marking light unit includes a marking light assembly and a beam splitter; the marking light emitted by the marking light assembly is reflected or transmitted by the beam splitter, and the marking light is focused on the target object through the lens assembly to form a marking pattern; the marking pattern passes through the lens assembly, is transmitted or reflected by the beam splitter and enters the image sensor, and a marking image of the target object is obtained on the image sensor; the microscopic imaging unit irradiates the target object with the imaging light source, and a microscopic image of the target object is obtained on the image sensor;

[0020] The distance between the target object and the lens assembly can be adjusted through the Z-axis adjustment assembly, and by judging whether there is an image of the marking image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object.

[0021] The distance between the target object and the lens assembly can be adjusted through the Z-axis adjustment assembly, and by judging the clarity of the image of the marking image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object.

[0022] The distance between the target object and the lens assembly can be adjusted through the Z-axis adjustment assembly, and by judging the light intensity of the image of the marking image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object.

[0023] The distance between the target object and the lens assembly can be adjusted through the Z-axis adjustment assembly, and by judging the diameter of the light spot of the image of the marking image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object.

[0024] The distance between the image sensor and the marking light assembly and the lens assembly can be adjusted through the Z-axis adjustment assembly, and by judging the presence or absence, clarity, light intensity, or diameter of the light spot of the image of the marking image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object.

[0025] In the above-mentioned marked light-assisted microscopic imaging device, the target object is a detection chip, and the interior of the detection chip includes a sample accommodation cavity; the Z-axis adjustment component adjusts the distance between the detection chip and the lens component, marks the marked image on the upper surface of the sample accommodation cavity, and obtains the position of the Z-axis adjustment component as D1; the Z-axis adjustment component adjusts the distance between the detection chip and the lens component, marks the marked image on the lower surface of the sample accommodation cavity, and obtains the position of the Z-axis adjustment component as D2; the distance between D2 and D1 is the height of the sample accommodation cavity.

[0026] In the above-mentioned marked light-assisted microscopic imaging device, the marked light component includes a laser emission device, and the marked light is a laser; or an LED light source, and the LED light source emits red light, green light, blue light or yellow light; or the light emitted by a light bulb.

[0027] In the above-mentioned marked light-assisted microscopic imaging device, the marked light component includes a marked light source and a perforated light shield, and there are more than 3 holes on the perforated light shield; the light emitted by the marked light source passes through the perforated light shield to form more than 3 light spots on the surface of the target object; the light spots are imaged on the image sensor; by judging the presence or absence or clarity of the marked image in the light spots, the perpendicularity between the surface of the target object and the optical path is judged.

[0028] In the above-mentioned marked light-assisted microscopic imaging device, during the emission of the marked light by the marked light component, the imaging light source is turned on or off.

[0029] It can be that the microscopic imaging unit irradiates the target object through the imaging light source, and during the acquisition of the microscopic image of the target object by the image sensor, the marked light component turns on or off the marked light.

[0030] It can be that the wavelength of the light emitted by the imaging light source is different from or partially the same as the wavelength of the marked light emitted by the marked light component.

[0031] A marked light-assisted microscopic imaging method, the marked light emitted by the marked light component is reflected or transmitted by a beam splitter, the marked light is focused on the target object through a lens component to form a marked pattern; the marked pattern passes through the lens component, is transmitted or reflected by the beam splitter and enters the image sensor, and a marked image of the target object is obtained on the image sensor; the microscopic imaging unit irradiates the target object through the imaging light source, and a microscopic image of the target object is obtained on the image sensor;

[0032] By adjusting the distance between the target object and the lens component, it can be judged whether the marked image is imaged on the image sensor, and whether the microscopic imaging component is correctly focused on the target object.

[0033] By adjusting the distance between the target object and the lens component, it can be judged the clarity of the marked image imaged on the image sensor, and whether the microscopic imaging component is correctly focused on the target object.

[0034] By adjusting the distance between the object to be detected and the lens assembly, the light intensity of the marked image formed on the image sensor can be judged, and whether the microscopic imaging assembly is correctly focused on the object to be detected can be judged.

[0035] By adjusting the distance between the object to be detected and the lens assembly, the diameter of the light spot of the marked image formed on the image sensor can be judged, and whether the microscopic imaging assembly is correctly focused on the object to be detected can be judged.

[0036] By adjusting the distances between the image sensor and the marked light assembly and the lens assembly, and by judging the presence or absence, clarity, light intensity, or light spot diameter of the image formed by the marked image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the object to be detected.

[0037] In the above-mentioned microscopic imaging method assisted by marked light, the object to be detected is a detection chip, and a sample accommodation cavity is included inside the detection chip; the distance between the detection chip and the lens assembly is adjusted, the marked image is marked on the upper surface of the sample accommodation cavity, and the position of the Z-axis adjustment assembly is obtained as D1; the distance between the detection chip and the lens assembly is adjusted, the marked image is marked on the lower surface of the sample accommodation cavity, and the position of the Z-axis adjustment assembly is obtained as D2; the distance between D2 and D1 is the height of the sample accommodation cavity.

[0038] In the above-mentioned microscopic imaging method assisted by marked light, the object to be detected is the upper surface or the lower surface of the sample accommodation cavity in the detection chip;

[0039] It can be that the object to be detected is the lower surface of the sample accommodation cavity in the detection chip. After the microscopic imaging assembly completes focusing on the lower surface of the sample accommodation cavity; the focusing plane is moved inside the cavity to obtain the focusing plane of the object to be detected, and the distance of moving the focusing plane is equal to the set value.

[0040] It can be that the object to be detected is the upper surface of the sample accommodation cavity in the detection chip. After the microscopic imaging assembly completes focusing on the upper surface of the sample accommodation cavity; the focusing plane is moved inside the cavity to obtain the focusing plane of the object to be detected, and the distance of moving the focusing plane is equal to the set value.

[0041] It can be that the object to be detected is the lower surface of the sample accommodation cavity in the detection chip. After the microscopic imaging assembly completes focusing on the lower surface of the sample accommodation cavity; the focusing plane is gradually moved inside the cavity to search for the object to be detected.

[0042] It can be that the object to be detected is the upper surface of the sample accommodation cavity in the detection chip. After the microscopic imaging assembly completes focusing on the upper surface of the sample accommodation cavity; the focusing plane is gradually moved inside the cavity to search for the object to be detected.

[0043] In the above-mentioned fluorescence-assisted microscopy imaging method, the imaging light source can be turned on or off during the emission of the fluorescence by the fluorescence component.

[0044] The above-mentioned microscopy imaging unit irradiates the target object with the imaging light source. During the acquisition of the microscopic image of the target object by the image sensor, the fluorescence component turns on or off the fluorescence.

[0045] The wavelength of the light emitted by the above-mentioned imaging light source is different from or partially the same as the wavelength of the fluorescence emitted by the fluorescence component. The above-mentioned microscopy imaging unit irradiates the target object with the imaging light source. During the acquisition of the microscopic image of the target object by the image sensor, the fluorescence component turns on the fluorescence, and the acquired microscopic image includes the fluorescence image and the microscopic image of the target object. The quality of the microscopic image of the target object is judged by the quality of the fluorescence image.

[0046] In the above-mentioned fluorescence-assisted microscopy imaging method, the detection chip contains a blood dilution sample.

[0047] The above-mentioned set value is equivalent to the radius of white blood cells. The above-mentioned set value is equivalent to the radius of red blood cells. The above-mentioned set value is equivalent to the radius of platelets.

[0048] The above-mentioned detection chip contains a urine sample or a urine concentrated sample or a urine diluted sample, and the target object is a cast, crystal, cell, and / or pathogenic microorganism.

[0049] In the above-mentioned fluorescence-assisted microscopy imaging method, the detection chip contains a fecal suspension, and the target object is a parasite egg, intestinal protozoa, starch granule, lipid droplet, plant fiber, muscle fiber, cell, and / or pathogenic microorganism.

[0050] In the above-mentioned fluorescence-assisted microscopy imaging method; the fluorescence image; is imaged on the image sensor;

[0051] By judging whether the fluorescence image shows or lacks parts, or whether there is any local clarity, the perpendicularity of the surface of the target object to the optical path is judged.

[0052] The technical effects of the above-mentioned technical solution include: analyzing whether the fluorescence is on the focal plane where focusing is desired, enabling accurate focusing, reducing the complexity of the focusing software, and basically not requiring complex focusing algorithms.

[0053] The technical effects of the above-mentioned technical solution include: converting the fuzzy algorithm focusing process into an accurate measurement process, and the focal plane adjustment approaches the theoretical focal plane position.

[0054] The technical effects of the above-mentioned technical solution include: whether the imaging light source is turned on or off does not affect the appearance of the light spot in the fluorescence image, but the focal length of the focusing is consistent with the imaging focus of the imaging light.

[0055] The technical effects of the above technical solution include: by adjusting the object distance, the focusing mark light is adjusted simultaneously. Through the change of the external focusing of the mark light, the cavity height of the test chip can be accurately measured, and the cavity height can be tested online.

[0056] The technical effects of the above technical solution include: by measuring the cavity height of the chip with a laser, the measurement accuracy is improved to more than 0.1 micrometer, and the measurement accuracy can be greatly improved.

[0057] The technical effects of the above technical solution include: according to the light intensity of the mark pattern obtained by focusing on different surfaces of the chip, the position of the current surface can be judged, and the initial position can be quickly determined.

[0058] The technical effects of the above technical solution include: through the laser, a stable mark pattern can be formed. Through the light intensity, spot size, and presence or absence of a spot, the focusing effect can be accurately judged, which is simple and efficient.

[0059] The technical effects of the above technical solution include: the mark pattern can be formed on the transparent upper and lower surfaces, so that it can be focused on the transparent surface, overcoming the problem that the traditional microscope cannot focus on transparent objects.

[0060] The technical effects of the above technical solution include: the mark pattern can be formed on the transparent upper and lower surfaces, so that it can be focused on the transparent surface, and the measurement reference for measuring cells or other targets can be found. Description of the Drawings

[0061] Figure 1 is a schematic diagram of the module division of a microscopic imaging device with mark light assistance;

[0062] Figure 2 is a schematic diagram of the imaging optical path of a microscopic imaging device with mark light assistance for a test chip;

[0063] Figure 3 is a schematic diagram of the imaging optical path for the upper surface of the cavity of the test chip;

[0064] Figure 4 is a schematic diagram of the imaging optical path for the lower surface of the cavity of the test chip;

[0065] Figure 5 is a schematic diagram of the imaging optical path for the lower surface of the test chip;

[0066] Figure 6 is a schematic diagram of different focusing focal planes of blood cells in the internal cavity of a test chip;

[0067] Figure 7 is a schematic diagram of the implementation of the mark light unit;

[0068] Figure 8 is a schematic diagram of the implementation of the mark pattern of the mark light;

[0069] Figure 9 It is a schematic diagram of microscopic imaging of red blood cells with marked light spots;;

[0070] Figure 10 It is a schematic diagram of an implementation scheme of a microscopic imaging device with marked light assistance;

[0071] Figure 11 It is a structural schematic diagram of a laser confocal microscope;

[0072] Figure 12 It is a schematic diagram of the optical path of a confocal microscope;

[0073] Figure 13 It is a schematic diagram of measuring the object distance of a microscope by a laser rangefinder. Specific implementation manners

[0074] The following further details the content of this application in conjunction with each attached drawing. It should be noted that the following is a description of the preferred embodiments of the present invention and does not constitute any limitation to the present invention. The description of the preferred embodiments of the present invention is only for the description of the general principle of the present invention. The numbers such as "first", "second", "A", and "B" involved in the present invention are only for the convenience of description and do not represent the sequence relationship in time or space. The combinations of letters and numbers "TA", "TB", and "H" involved in the present invention are only for the convenience of description, and the specific meanings are determined by the specific words they represent.

[0075] Such as Figure 1, A marker light-assisted microscopic imaging device, comprising a microscopic imaging unit and a marker light unit; the microscopic imaging unit includes an image sensor, a lens assembly, an imaging light source, and a Z-axis adjustment assembly; the marker light unit includes a marker light assembly and a beam splitter; the marker light emitted by the marker light assembly is reflected or transmitted by the beam splitter, and the marker light is focused on the target object through the lens assembly to form a marker pattern; the marker pattern passes through the lens assembly, is transmitted or reflected by the beam splitter and enters the image sensor, and a marker image of the target object is obtained on the image sensor; the microscopic imaging unit irradiates the target object with the imaging light source, and a microscopic image of the target object is obtained on the image sensor; the distance between the target object and the lens assembly can be adjusted by the Z-axis adjustment assembly, and by judging whether there is an image of the marker image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object. The distance between the target object and the lens assembly can be adjusted by the Z-axis adjustment assembly, and by judging the clarity of the marker image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object. The distance between the target object and the lens assembly can be adjusted by the Z-axis adjustment assembly, and by judging the light intensity of the marker image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object. The distance between the target object and the lens assembly can be adjusted by the Z-axis adjustment assembly, and by judging the diameter of the light spot of the marker image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object. The distance between the image sensor and the marker light assembly and the lens assembly can be adjusted by the Z-axis adjustment assembly, and by judging the presence or absence, clarity, light intensity, or diameter of the light spot of the marker image on the image sensor, it can be judged whether the microscopic imaging assembly is correctly focused on the target object.

[0076] Image sensors mainly include two categories: CCD (Charge-Coupled Device) and CMOS (Complementary Metal-Oxide Semiconductor).

[0077] Modern digital microscopes generally integrate a digital camera with complete functions. Generally, digital cameras have an autofocus function. However, in the usage scenario of accurately measuring the size of microscopic objects, the autofocus function is difficult to align with the target object. Or after the digital camera adjusts the focal length, it is difficult to obtain the size of microscopic objects through calculation. In many scenarios, it is necessary to lock the magnification and focus by adjusting the object distance.

[0078] By observing with the human eye or an image analysis component electrically connected to the image sensor, it can be analyzed whether the marker light is on the desired focal plane, and accurate focusing can be achieved.

[0079] Such as Figure 2, The marking light emitted by the marking light component will converge into a point through the lens component. By setting the positions of the marking light component and the image sensor, the convergence point and the focal plane can be adjusted to the same plane. When the Z-axis adjustment component is adjusted to drive the convergence point to move up and down, when the convergence point reaches the target object, a clear image can be obtained on the image sensor. After passing through the lens component, when observing the target object with a microscope, there is always a depth of field. The smaller the depth of field, the more coincident the focal plane and the convergence point are.

[0080] The principle of the marking pattern emitted by the marking light component is the same. The light-emitting point of the marking light is a dot pattern.

[0081] Generally, the light beam diameter of a laser is 1 mm. After passing through the lens component, it can be converged into a very small point, which can penetrate multiple layers of glass or transparent materials and converge into a light spot on the glass surface. The light spot reflects or diffuses, and can form a marking image on the image sensor.

[0082] Such as Figure 2 , 3 , 4, 5, for the above-mentioned marking light-assisted microscopic imaging device, the target object is a detection chip, and the detection chip internally includes a sample accommodation cavity; the Z-axis adjustment component adjusts the distance between the detection chip and the lens component, marks the marking image on the upper surface of the sample accommodation cavity, and obtains the position of the Z-axis adjustment component as D1; the Z-axis adjustment component adjusts the distance between the detection chip and the lens component, marks the marking image on the lower surface of the sample accommodation cavity, and obtains the position of the Z-axis adjustment component as D2; the distance between D2 and D1 is the height of the sample accommodation cavity.

[0083] Figure 2 , The marking light is projected onto the upper surface of the detection chip, and the focal plane of the lens group also corresponds to the upper surface of the detection chip. Due to the reflection and diffuse reflection on the upper surface, the marking light will form a relatively bright light spot. In the marking image obtained by the image sensor, a brightest light spot or the brightest image will be obtained. At this time, whether the imaging light source is turned on or off does not affect the appearance of the light spot in the marking image.

[0084] Figure 3 , When the distance between the detection chip and the lens group is reduced, that is, the distance from the objective lens, the marking light is projected onto the upper surface of the detection cavity, and the focal plane of the lens group also corresponds to the upper surface of the detection cavity. In the marking image obtained by the image sensor, a relatively bright light spot or image will be obtained. At this time, the position of the Z-axis adjustment component is D1.

[0085] Figure 4 , When the distance between the detection chip and the lens group is further reduced, the marking light is projected onto the lower surface of the detection cavity, and the focal plane of the lens group also corresponds to the lower surface of the detection cavity. In the marking image obtained by the image sensor, a relatively dim light spot or image will be obtained. At this time, the position of the Z-axis adjustment component is D2.

[0086] Calculate the distance between D1 and D2, which is the height of the cavity. The measurement can be done before or after microscopic imaging. With the accurate height of the measured cavity, and based on the pixel area and magnification factor corresponding to the obtained image, the area of the measured liquid can be obtained. According to the area and the height value of the cavity, the volume value of the measured liquid can be obtained.

[0087] Figure 4 , continue to move the detection chip closer to the lens group. The marking light is projected onto the lower surface of the detection chip, and the focal plane of the lens group also corresponds to the lower surface of the detection chip. In the marking image obtained by the image sensor, a dark light spot or image will be obtained. Through the intensity of the marking light on different surfaces, the image analysis component can know the current original position height and can enter the working state more quickly.

[0088] Such as Figure 6 , the detection chip includes two layers of transparent materials on the upper and lower sides, and a cavity for accommodating the detection liquid in the middle. If a blood sample is accommodated, white blood cells, red blood cells, and platelets in the blood will sink to the bottom. By finding the lower surface of the detection cavity, that is, finding the focal plane D0. If it is a blood sample, by moving up the radius height of the platelets, the focal plane is adjusted to position D1. At this time, the image of the platelets can be observed; then move the focal plane up to the radius height of the red blood cells, and the focal plane can be adjusted to position D2, and the image of the red blood cells can be observed; then move the focal plane up to the radius height of the white blood cells, and the focal plane can be adjusted to position D3, and the image of the white blood cells can be observed. The number of white blood cells is small, especially basophils, and there may be none in one field of view of the microscope. By horizontally moving at the position of the focal plane D3, the target can be searched for in multiple images.

[0089] For the above-mentioned marking light-assisted microscopic imaging device, the marking light component includes a laser emitting device, and the marking light is a laser; or an LED light source, and the LED light source emits red light, green light, blue light, or yellow light; or the light emitted by a light bulb.

[0090] Such as Figure 7 , for the above-mentioned marking light-assisted microscopic imaging device, the marking light component includes a marking light source and a perforated light-shielding plate, and there are more than 3 holes on the perforated light-shielding plate; the light emitted by the marking light source passes through the perforated light-shielding plate to form more than 3 light spots on the surface of the target; the light spots are imaged on the image sensor; by judging the presence or absence or clarity of the marking image on the light spots, the perpendicularity of the surface of the target to the optical path is judged.

[0091] Microscopic imaging, the perpendicularity between the detection chip and the microscopic imaging unit is closely related to target search and measurement. If it is not perpendicular, due to the depth of field, in one image, the target objects at many positions may not be imaged and observed, or it may cause some imaged cells to be large and some to be small.

[0092] For example Figure 8 , the perforated light shield has 5 holes. If the surface of the target object is not perpendicular to the optical path, the imaging states of the 4 peripheral holes will be inconsistent, and it can be adjusted by a device for adjusting perpendicularity to make the imaging states of the 4 holes consistent.

[0093] For example Figure 9 , if the marker light group is turned on during microscopic imaging, an imaging diagram of blood cells with light spots can be obtained, and by judging the shape of the light spots, it can be determined whether the picture is qualified.

[0094] In the above-mentioned microscopic imaging device with marker light assistance, during the emission of the marker light by the marker light component, the imaging light source is turned on or off.

[0095] It can be that the microscopic imaging unit irradiates the target object with the imaging light source, and during the acquisition of the microscopic image of the target object by the image sensor, the marker light component turns on or off the marker light.

[0096] It can be that the wavelength of the light emitted by the imaging light source is different from or partially the same as the wavelength of the marker light emitted by the marker light component.

[0097] When the wavelengths of the imaging light source and the marker light component are different, the marker light can be distinguished in the image, and lights with different wavelengths also have a detection function when detecting the target, such as purple light and infrared light. That is, the marker light can also be used as a measurement light in some measurement scenarios.

[0098] For example Figure 10 , C1 can be the image sensor in the camera or an assembled image sensor, PL is the marker light component, which can be a point light source device, a laser device or an LED panel. L2’ corresponds to the object distance of the marker light S1’, L3’ corresponds to the image distance of S1’, L3’ = (L21 + L22), H, H’ are the reference planes, the object-side principal plane and the image-side principal plane, and the position of the marker light component PL can be swapped with the position of the camera C1.

[0099] The position of the marker light component PL can be swapped with the position of the camera C1, that is, the marker light reaches the target point by transmission or reflection through the beam splitter. Also, the marker pattern passes through the beam splitter by transmission or reflection.

[0100] A method for marker light-assisted microscopy imaging. The marker light component emits marker light, which is reflected or transmitted by a beam splitter. The marker light is focused on the target object through a lens component to form a marker pattern. The marker pattern passes through the lens component, is transmitted or reflected by the beam splitter, and enters an image sensor, where a marker image of the target object is obtained. The microscopy imaging unit irradiates the target object with an imaging light source, and a microscopy image of the target object is obtained on the image sensor.

[0101] By adjusting the distance between the target object and the lens component, it is possible to determine whether the marker image is formed on the image sensor and whether the microscopy imaging component is correctly focused on the target object.

[0102] If the focal plane is far from the target object, the marker pattern will not be formed on the image sensor, and the marker image cannot be obtained.

[0103] By adjusting the distance between the target object and the lens component, it is possible to determine the clarity of the marker image formed on the image sensor and whether the microscopy imaging component is correctly focused on the target object.

[0104] If the focal plane is close to the target object, the marker pattern will be formed on the image sensor, and the clarity can be used to determine whether the focus is correct.

[0105] By adjusting the distance between the target object and the lens component, it is possible to determine the light intensity of the marker image formed on the image sensor and whether the microscopy imaging component is correctly focused on the target object.

[0106] If the focus between the focal plane and the target object is more accurate, the light intensity of the marker pattern is stronger. Of course, it is necessary to adjust L3’=(L21 + L22) in advance so that the imaging focal plane coincides with the convergence point of the marker light.

[0107] By adjusting the distance between the target object and the lens component, it is possible to determine the diameter of the light spot of the marker image formed on the image sensor and whether the microscopy imaging component is correctly focused on the target object.

[0108] If a laser is used as the marker light, after passing through the lens, the beam diameter will become smaller. By making the imaging focal plane coincide with the convergence point of the marker light in advance, the light spot diameter is the smallest at this time. By measuring the size of the light spot diameter, the quality of the focus can be judged.

[0109] By adjusting the distance between the image sensor and the marker light component and the lens component, and by judging the presence or absence, clarity, light intensity, or the diameter of the light spot of the marker image formed on the image sensor, it is possible to determine whether the microscopy imaging component is correctly focused on the target object.

[0110] Adjusting the distance between the image sensor and the marker light assembly and the lens assembly is equivalent to adjusting the object distance. At this time, the object distance will also change, and accurate focusing can be obtained. However, in this case, the magnification will also change, resulting in complex quantitative calculations. In scenarios where precise measurement is not required, this method can be used to quickly obtain focus.

[0111] In the above-mentioned marker light-assisted microscopic imaging method, the target object is a detection chip, and the detection chip includes a sample accommodation cavity inside; adjusting the distance between the detection chip and the lens assembly, marking the marker image on the upper surface of the sample accommodation cavity, and obtaining the position of the Z-axis adjustment assembly as D1; adjusting the distance between the detection chip and the lens assembly, marking the marker image on the lower surface of the sample accommodation cavity, and obtaining the position of the Z-axis adjustment assembly as D2; the distance between D2 and D1 is the height of the sample accommodation cavity.

[0112] In the above-mentioned marker light-assisted microscopic imaging method, the target object is the upper surface or the lower surface of the sample accommodation cavity in the detection chip;

[0113] It can be that the target object is the lower surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the lower surface of the sample accommodation cavity; moving the focus plane inward into the cavity to obtain the focus plane of the object to be detected, and the distance of moving the focus plane is equal to the set value.

[0114] For different target objects, there is a relatively fixed size range. After finding the reference focus plane, it can quickly move to the focus plane of the target object.

[0115] It can be that the target object is the upper surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the upper surface of the sample accommodation cavity; moving the focus plane inward into the cavity to obtain the focus plane of the object to be detected, and the distance of moving the focus plane is equal to the set value.

[0116] It can be that the target object is the lower surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the lower surface of the sample accommodation cavity; gradually moving the focus plane inward into the cavity to search for the detection target object.

[0117] It can be that the target object is the upper surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the upper surface of the sample accommodation cavity; gradually moving the focus plane inward into the cavity to search for the detection target object.

[0118] In the above-mentioned marker light-assisted microscopic imaging method, it can be that during the emission of the marker light by the marker light assembly, the imaging light source is turned on or off.

[0119] The above-mentioned microscopic imaging unit irradiates the target object with the imaging light source. During the acquisition of the microscopic image of the target object by the image sensor, the marker light assembly turns on or off the marker light.

[0120] The wavelength of the light emitted by the above-mentioned imaging light source is not the same as or partially the same as the wavelength of the marking light emitted by the marking light assembly. The above-mentioned microscopic imaging unit irradiates the target object with the imaging light source. During the acquisition of the microscopic image of the target object by the image sensor, the marking light assembly turns on the marking light. The obtained microscopic image includes the marking image and the microscopic image of the target object. The quality of the microscopic image of the target object is judged by the quality of the marking image.

[0121] In the above-mentioned microscopic imaging method assisted by marking light, the detection chip contains a blood dilution sample.

[0122] The above-mentioned set value is equivalent to the radius of white blood cells. The above-mentioned set value is equivalent to the radius of red blood cells. The above-mentioned set value is equivalent to the radius of platelets.

[0123] The above-mentioned detection chip contains a urine sample or a urine concentrate sample or a urine dilution sample, and the target object is a cast, crystal, cell, and / or pathogenic microorganism.

[0124] In the above-mentioned microscopic imaging method assisted by marking light, the detection chip contains a fecal suspension, and the target object is a parasite egg, intestinal protozoa, starch granule, lipid droplet, plant fiber, muscle fiber, cell, and / or pathogenic microorganism.

[0125] In the above-mentioned microscopic imaging method assisted by marking light; the above-mentioned marking image; is imaged on the image sensor;

[0126] By judging whether the marking image shows or lacks parts, or the presence or absence or clarity of a local area, the perpendicularity of the surface of the target object to the optical path is judged.

[0127] Although the present invention is described and illustrated according to preferred embodiments and several alternative solutions, the invention is not limited by the specific descriptions in this specification. Other additional alternatives or equivalent components can also be used to practice the present invention.

Claims

1. A marked light-assisted microscopic imaging device, characterized in that It includes a microscopic imaging unit and a marking light unit; The microscopic imaging unit includes an image sensor, a lens assembly, an imaging light source, and a Z-axis adjustment assembly; The marking light unit includes a marking light assembly and a beam splitter; The marking light assembly emits marking light that is reflected or transmitted by the beam splitter. The marking light is focused on the target object through the lens assembly to form a marking pattern; The marking pattern passes through the lens assembly, is transmitted or reflected by the beam splitter, and enters the image sensor, and a marking image of the target object is obtained on the image sensor; The microscopic imaging unit irradiates the target object with the imaging light source, and a microscopic image of the target object is obtained on the image sensor; it includes any one of the following technical features, Feature TA10: The Z-axis adjustment assembly adjusts the distance between the target object and the lens assembly, and judges whether the microscopic imaging assembly is correctly focused on the target object by judging the presence or absence of the marking image on the image sensor; Feature TA20: The Z-axis adjustment assembly adjusts the distance between the target object and the lens assembly, and judges whether the microscopic imaging assembly is correctly focused on the target object by judging the clarity of the marking image on the image sensor; Feature TA30: The Z-axis adjustment assembly adjusts the distance between the target object and the lens assembly, and judges whether the microscopic imaging assembly is correctly focused on the target object by judging the light intensity of the marking image on the image sensor; Feature TA40: The Z-axis adjustment assembly adjusts the distance between the target object and the lens assembly, and judges whether the microscopic imaging assembly is correctly focused on the target object by judging the diameter of the light spot of the marking image on the image sensor; Feature TA50: The Z-axis adjustment assembly adjusts the distance between the image sensor and the marking light assembly and the lens assembly, and judges whether the microscopic imaging assembly is correctly focused on the target object by judging the presence or absence, clarity, light intensity, or diameter of the light spot of the marking image on the image sensor.

2. The marked light-assisted microscopic imaging device according to claim 1, wherein The target object is a detection chip, and a sample accommodation cavity is included inside the detection chip; The Z-axis adjustment assembly adjusts the distance between the detection chip and the lens assembly, marks the marking image on the upper surface of the sample accommodation cavity, and obtains the position of the Z-axis adjustment assembly as D1; The Z-axis adjustment assembly adjusts the distance between the detection chip and the lens assembly, marks the marking image on the lower surface of the sample accommodation cavity, and obtains the position of the Z-axis adjustment assembly as D2; The distance between D2 and D1 is the height of the sample accommodation cavity.

3. The labeled light-assisted microscopic imaging device according to claim 1, wherein The marking light assembly includes a laser emitting device, and the marking light is laser; or an LED light source, and the LED light source emits red light, green light, blue light, or yellow light; or the light emitted by a bulb.

4. The labeled light-assisted microscopic imaging device according to claim 1, characterized in that, The marking light assembly includes a marking light source and a perforated light shield, and there are more than 3 holes on the perforated light shield; The light emitted by the marking light source passes through the perforated light shield to form more than 3 light spots on the surface of the target object; The light spots are imaged on the image sensor; By judging the presence or absence or clarity of the marking image on the light spots, the perpendicularity between the surface of the target object and the optical path is judged.

5. The marking light-assisted microscopic imaging device according to claim 1, wherein, It includes any one of the following technical features, Feature TB10: During the emission of the marking light by the marking light component in the feature TA10 or feature TA20, the imaging light source is turned on or off; Feature TB20: The microscopic imaging unit irradiates the target object with the imaging light source, and during the acquisition of the microscopic image of the target object by the image sensor, the marking light component turns on or off the marking light; Feature TB30: The wavelength of the light emitted by the imaging light source is different from or partially the same as the wavelength of the marking light emitted by the marking light component.

6. A microscopic imaging method with marking light assistance, characterized in that, The marking light emitted by the marking light component is reflected or transmitted by the beam splitter, and the marking light is focused on the target object through the lens assembly to form a marking pattern; The marking pattern passes through the lens assembly, is transmitted or reflected by the beam splitter and enters the image sensor, and a marking image of the target object is obtained by the image sensor; The microscopic imaging unit irradiates the target object with the imaging light source, and a microscopic image of the target object is obtained by the image sensor; including any one of the following technical features, Feature TC10: Adjust the distance between the target object and the lens assembly, and judge whether the microscopic imaging component is correctly focused on the target object by judging the presence or absence of the marking image on the image sensor; Feature TC20: Adjust the distance between the target object and the lens assembly, and judge whether the microscopic imaging component is correctly focused on the target object by judging the clarity of the marking image on the image sensor; Feature TC30: Adjust the distance between the target object and the lens assembly, and judge whether the microscopic imaging component is correctly focused on the target object by judging the light intensity of the marking image on the image sensor; Feature TC40: Adjust the distance between the target object and the lens assembly, and judge whether the microscopic imaging component is correctly focused on the target object by judging the diameter of the light spot of the marking image on the image sensor; Feature TC50: Adjust the distance between the image sensor and the marking light component and the lens assembly, and judge whether the microscopic imaging component is correctly focused on the target object by judging the presence or absence or clarity or light intensity or the diameter of the light spot of the marking image on the image sensor.

7. The method for marked light-assisted microscopic imaging according to claim 6, wherein The target object is a detection chip, and the detection chip internally includes a sample accommodation cavity; Adjust the distance between the detection chip and the lens assembly, mark the marking image on the upper surface of the sample accommodation cavity, and obtain the position of the Z-axis adjustment component as D1; Adjust the distance between the detection chip and the lens assembly, mark the marking image on the lower surface of the sample accommodation cavity, and obtain the position of the Z-axis adjustment component as D2; The distance between D2 and D1 is the height of the sample accommodation cavity.

8. The marked light-assisted microscopic imaging method according to claim 6, wherein The target object is the upper surface or the lower surface of the sample accommodation cavity in the detection chip; Including any one of the following technical features, Feature TD10: The target object is the lower surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the lower surface of the sample accommodation cavity; move the focusing plane inward into the cavity to obtain the focusing plane of the object to be detected, and the distance of moving the focusing plane is equal to the set value; Feature TD20: The target is the upper surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the upper surface of the sample accommodation cavity, move the focus plane inward into the cavity to obtain the focus plane of the object to be detected, and the distance of moving the focus plane is equal to the set value; Feature TD30: The target is the lower surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the lower surface of the sample accommodation cavity, gradually move the focus plane inward into the cavity to search for the detection target; Feature TD40: The target is the upper surface of the sample accommodation cavity in the detection chip. After the microscopic imaging component completes focusing on the upper surface of the sample accommodation cavity, gradually move the focus plane inward into the cavity to search for the detection target.

9. The method for marker light-assisted microscopic imaging according to claim 6, characterized in that It includes any one of the following technical features Feature TE10: During the emission of the marker light by the marker light component, the imaging light source is turned on or off; Feature TE20: The microscopic imaging unit irradiates the target with the imaging light source. During the acquisition of the microscopic image of the target by the image sensor, the marker light component turns on or off the marker light; Feature TE30: The wavelength of the light emitted by the imaging light source is different from or partially the same as the wavelength of the marker light emitted by the marker light component; Feature TE40: The microscopic imaging unit irradiates the target with the imaging light source. During the acquisition of the microscopic image of the target by the image sensor, the marker light component turns on the marker light, and the obtained microscopic image includes the marker image and the microscopic image of the target. The quality of the microscopic image of the target is judged by the quality of the marker image.

10. The method for labeled light-assisted microscopy imaging according to claim 8, characterized in that, The detection chip contains a blood dilution sample, including any one of the following technical features Feature TF10: The set value is equivalent to the radius of a white blood cell; Feature TF10: The set value is equivalent to the radius of a red blood cell; Feature TF10: The set value is equivalent to the radius of a platelet.

11. The marked light-assisted microscopic imaging method according to claim 8, wherein The detection chip contains a urine sample or a urine concentrate sample or a urine dilution sample, and the target is a cast, crystal, cell, and / or pathogenic microorganism.

12. The method for marked light-assisted microscopic imaging according to claim 8, wherein The detection chip contains a fecal suspension, and the target is a parasite egg, intestinal protozoa, starch granule, lipid droplet, plant fiber, muscle fiber, cell, and / or pathogenic microorganism.

13. The marked light-assisted microscopic imaging method according to claim 6, wherein The marker image; Is imaged on the image sensor; Judge the perpendicularity of the surface of the target to the optical path by judging whether the marker image shows or lacks parts, or the presence or absence or clarity of a local area.

Images