CN117147551B - Chromosome scanning imaging method and device - Google Patents
Chromosome scanning imaging method and device Download PDFInfo
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Abstract
The invention discloses a chromosome scanning imaging method, which belongs to the technical field of microscopic imaging and comprises the following steps: loading a sample; preliminary focusing search is carried out on the low-power objective lens; low power objective FPM reconstruction; high-power objective lens precise focusing; the method comprises the steps of reconstructing a high-power objective FPM, scanning and imaging a chromosome by adopting a Fourier microscopic laminated imaging technology, wherein in the low-power searching process, the field of view of the objective is larger, and the number of fields of view of full-wave chip searching is greatly reduced; in the high-power final imaging process, the method is determined according to X, Y coordinates of each metaphase chromosome identified after low-power reconstruction, and only the position of the metaphase chromosome is reconstructed, so that the high-resolution imaging without an oil mirror is realized, and various defects caused by an automatic oil dripping system are avoided. The application also provides a device for implementing the chromosome scanning imaging method.
Description
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to a chromosome scanning imaging method and a device for implementing the chromosome scanning imaging method.
Background
In human chromosome karyotyping processIn the course of which microscopic imaging of chromosomes is required using an optical microscope. During imaging, a low power objective lens (typically 10 × Objective lens, numerical aperture NA 0.25 or more), searching for metaphase chromosome image in the whole sample slide range, and passing through high power objective lens (generally 100) × Oil mirror, numerical aperture NA is more than or equal to 1.25) to more accurately focus the searched image to obtain a clear chromosome image for subsequent karyotype analysis. In one aspect, 10 × The field of view of the objective lens is still not large enough, full slide search is realized, imaging with tens or hundreds of fields of view is needed, the scanning time is long, the efficiency is low, and the imaging effect of the objective lens with larger field of view and lower power is insufficient for completing mid-stage chromosome search work with high accuracy because of smaller NA value; on the other hand, in the full-automatic high-throughput chromosome scanning imaging equipment with extremely high requirement on the current automation degree, frequent automatic oil dripping can increase the whole working time of the instrument, reduce the comprehensive efficiency of scanning, and the microscope oil is viscous and difficult to clean, so that the long-time storage and multiplexing of subsequent samples are not facilitated.
Disclosure of Invention
Aiming at the defects existing in the prior art, one of the main purposes of the invention is to provide a chromosome scanning imaging method for realizing low-power objective high-flux scanning and simultaneously realizing oil-free imaging.
Aiming at the defects existing in the prior art, the second main object of the invention is to provide a chromosome scanning imaging device which realizes high-flux scanning of a low-power objective lens and simultaneously performs oil-free imaging.
One of the purposes of the invention is realized by adopting the following technical scheme:
a chromosome scanning imaging method comprising the steps of:
loading a sample: placing the slide with the chromosome sample smeared thereon on the stage assembly;
preliminary focusing search of a low-power objective lens: focusing the glass slide in each view through the low-power objective lens, and recording Z-position coordinates corresponding to the low-power objective lens;
low power objective FPM reconstruction: performing Fourier stacked microscopic imaging on each view of the low power objective lens, judging chromosomes in each view to identify metaphase chromosomes, and recording X, Y position coordinates of the metaphase chromosomes;
high power objective lens precise focusing: according to the Z-position coordinates of the preliminary focusing of the low-power objective lens, carrying out precise focusing on the glass slide in each view through the high-power objective lens;
high power objective FPM reconstruction: fourier stacked microscopy imaging is performed on each field of view of the high power objective lens according to metaphase chromosome X, Y position coordinates to obtain a high resolution image.
Further, the low power objective FPM reconstruction specifically includes the following steps:
illuminating M LED light sources which take a central LED as a symmetrical center on the LED flat light sources one by one with first brightness to illuminate the glass slide in the visual field;
the camera shoots M low-resolution images in sequence;
initializing a low resolution image;
performing FPM reconstruction iteration;
obtaining a high-resolution image;
judging whether a metaphase chromosome exists in the high-resolution image, if so, recording X, Y position coordinates of the metaphase chromosome; if not, the low power objective lens performs preliminary focusing search on the glass slide in the next view.
Further, the FPM reconstruction iteration specifically includes the following steps:
carrying out synthetic aperture operation on the low-resolution image one by one in a frequency domain by adopting an iterative method based on the combination of a synthetic aperture technology and a phase recovery algorithm;
and stopping the iterative process when the cost function value is smaller than a given threshold value by taking the cost function value as a criterion, and taking the amplitude and the phase of the high-resolution image obtained at the moment as the high-resolution image.
Furthermore, the low-power objective FPM reconstruction further comprises an image denoising processing step, wherein the image denoising processing step is positioned between the steps of shooting M low-resolution images in sequence by the camera and initializing the low-resolution images.
Further, the high power objective FPM reconstruction specifically includes the following steps:
illuminating the glass slides in the visual field by illuminating N LED light sources on the LED flat light sources one by one with the second brightness, wherein the N LED light sources take a central LED as a symmetrical center;
the camera shoots N low-resolution images in sequence;
initializing a low resolution image;
performing FPM reconstruction iteration;
a high resolution image is obtained.
Further, the second luminance is higher than the first luminance, and N < M.
Further, the preliminary focusing search of the low power objective lens specifically includes the following steps:
illuminating an LED flat light source to illuminate the slide;
the control system component drives the low-power objective lens to move along the Z direction, and chromosome images are obtained through the automatic focusing component and the camera;
after the imaging is clear, focusing is stopped, and Z-position coordinates of the low-power objective lens are recorded.
Further, the loading sample specifically includes the following steps:
sequentially placing a plurality of slides smeared with chromosome samples on a slide yoke plate;
placing a plurality of slide yoke plates in a containing cavity of a storage box from bottom to top;
driving the storage box to move to a designated position;
a slide linkage is loaded into the recess in the stage assembly by the automatic catch assembly.
Further, the method further comprises an objective lens switching step, wherein the objective lens switching step is between the low-power objective lens FPM reconstruction step and the high-power objective lens precise focusing step, and specifically comprises the following steps: the objective turntable is driven to rotate around the axis of the objective seat by the objective switching transmission component, so that the switching action of the low-power objective and the high-power objective is realized.
The second purpose of the invention is realized by adopting the following technical scheme:
the chromosome scanning imaging device is used for implementing the chromosome scanning imaging method of any one of the above, and comprises a base, a microscopic imaging component, a stage component and a control system component, wherein the stage component is movably arranged on the base, a glass slide is placed on the stage component, the microscopic imaging component comprises an objective lens switching component and is movably arranged on the base, the objective lens switching component comprises a low-power objective lens and a high-power objective lens, the low-power objective lens is used for carrying out preliminary focusing on the glass slide in a single view field, the control system component controls the high-power objective lens to carry out precise focusing on each view field according to Z-position coordinates of the preliminary focusing of the low-power objective lens, and the control system component identifies and positions a middle-stage chromosome in each view field after Fourier laminated microscopic imaging of the low-power objective lens, and the high-power objective lens carries out Fourier laminated microscopic imaging according to the position coordinates of the middle-stage chromosome X, Y so as to acquire a high-resolution image.
Further, the microscopic imaging assembly further comprises an LED array illumination assembly, the LED array illumination assembly comprises a light source support, a two-dimensional manual adjustment table and an LED panel light source, the light source support is installed on the base, the two-dimensional manual adjustment table is installed on the light source support and used for manually fine adjusting the position of the light source, the LED panel light source is movably installed on the table top of the two-dimensional manual adjustment table and is in communication connection with the control system assembly, and the control system assembly adjusts the luminous brightness and the luminous quantity of the LED panel light source according to the low-power objective lens or the high-power objective lens.
Further, the objective table component comprises an X-direction transmission component, a Y-direction transmission component, an objective table lower plate, an objective table middle plate and an objective table upper plate, wherein the objective table lower plate is fixedly arranged on the base, the objective table middle plate is movably arranged on the objective table lower plate, and the Y-direction transmission component drives the objective table middle plate to perform scanning motion along the Y direction; the object stage upper plate is movably arranged on the object stage middle plate, and the X-direction transmission component drives the object stage upper plate to perform scanning motion along the X direction.
Further, the microscopic imaging assembly further comprises a microscope frame and an automatic focusing assembly, the microscope frame is mounted on the base, the automatic focusing assembly comprises a focusing transmission assembly and a focusing lifting seat, the focusing lifting seat is mounted on the microscope frame and is in transmission connection with the focusing transmission assembly, the objective lens switching assembly is arranged on the focusing lifting seat, and the focusing transmission assembly drives the focusing lifting seat to move so as to drive the low-power objective lens or the high-power objective lens to focus along Z-direction movement.
Compared with the prior art, the chromosome scanning imaging method has the following beneficial effects:
(1) The invention adopts the Fourier microscopic lamination imaging technology to scan and image the chromosome, in the process of low-power searching, the field of view of the objective lens is larger, the number of the fields of view of the whole glass slide searching is greatly reduced, and the scanning imaging flux of the chromosome low-power searching is obviously improved; in the high-power final imaging process, the method is determined according to X, Y coordinates of each metaphase chromosome identified after low-power reconstruction, and only the position of the metaphase chromosome is reconstructed, so that the high-resolution imaging without an oil mirror is realized, and various defects caused by an automatic oil dripping system are avoided.
(2) The invention adopts the LED array light source component with adjustable brightness to replace components such as a light source, a condenser lens, an electric diaphragm and the like in the traditional full-automatic microscope, can respectively adapt to the illumination requirements of low-power and high-power objective lenses, and reduces the complexity of a mechanical structure and a motion control unit in an illumination system.
(3) In the invention, even if the position coordinates of the object stage have deviation of a few micrometers or even tens of micrometers, the middle-stage chromosome can still appear in the visual field of the target surface of the camera, and in the process of low-power searching, the requirement on the scanning speed of the object stage is obviously reduced because the single visual field area is greatly improved.
(4) The invention greatly reduces the requirements of positioning precision, running speed and feeding resolution of a plurality of core modules of the automatic chromosome scanning imaging device, and can greatly reduce the development cost and difficulty of the system.
Drawings
For a clearer description of an embodiment of the invention, reference will be made to the accompanying drawings of embodiments, which are given for clarity, wherein:
FIG. 1 is a flow chart of the steps of a chromosome scanning imaging method of the present invention;
FIG. 2 is a flow chart of the steps of a low power objective FPM reconstruction of the chromosome scanning imaging method of FIG. 1;
FIG. 3-a is a chromosomal image obtained after a low power objective lens search in a conventional microscopy scanning imaging method;
FIG. 3-b is a chromosome image obtained after reconstruction of a low power objective FPM in a chromosome scanning imaging method of the present invention;
FIG. 4 is a flow chart of the steps of high power objective FPM reconstruction of the chromosome scanning imaging method of FIG. 1;
FIG. 5-a is a high resolution image obtained after imaging with a high magnification microscope in a conventional microscopy scanning imaging method;
FIG. 5-b is a high resolution image obtained after reconstruction of a high power objective FPM in a chromosome scanning imaging method of the present invention;
FIG. 6 is a perspective view of a chromosome scanning imaging apparatus of the present invention;
FIG. 7 is a schematic diagram of the microscopic imaging assembly of the chromosome scanning imaging apparatus of FIG. 6;
FIG. 8 is a schematic view of a partial structure of a microscopic imaging assembly of the chromosome scanning imaging apparatus of FIG. 7;
FIG. 9 is another partial schematic view of a microscopic imaging assembly of the chromosome scanning imaging apparatus of FIG. 7;
FIG. 10 is a schematic view of the stage assembly of the chromosome scanning imaging apparatus of FIG. 6;
FIG. 11 is another schematic structural view of a stage assembly of the chromosome scanning imaging apparatus of FIG. 6;
FIG. 12 is a schematic view of a magazine assembly of the chromosome scanning imaging apparatus of FIG. 6;
FIG. 13 is a schematic diagram of the control system components of the chromosome scanning imaging apparatus of FIG. 6;
FIG. 14 is a schematic diagram of the control circuitry of the chromosome scanning imaging apparatus of FIG. 13 in operation;
FIG. 15 is a schematic flow chart of the chromosome scanning imaging apparatus of the present invention in operation.
In the figure: 10. a base;
20. a microimaging assembly;
21. a microscope frame;
22. an auto-focus assembly; 221. a focusing transmission assembly; 222. a focusing transmission guide rail; 223. a focusing lifting seat; 224. a precision grating ruler assembly; 225. focusing photoelectric switch;
23. an objective lens switching assembly; 231. an objective lens switching transmission assembly; 232. an objective lens holder; 233. an objective lens switches the photoelectric switch; 234. a low power objective lens; 235. a high power objective lens; 236. an objective lens turntable;
24. an optical imaging assembly; 241. an imaging lens; 242. a transfer cylinder; 243. a camera;
25. an LED array illumination assembly; 251. a light source support; 252. a two-dimensional manual adjustment table; 253. an LED flat light source;
30. a stage assembly; 31. an X-direction transmission assembly; 32. a Y-direction transmission assembly; 33. a stage lower plate; 34. an objective table middle plate; 35. an objective table upper plate; 36. an X-ray guide rail assembly; 37. a Y-rail assembly; 38. a scanning table photoelectric switch;
40. an automatic hooking component; 41. a hook piece transmission assembly; 42. a hook piece guide rail component; 43. hook sheet metal; 44. a T-shaped hook;
50. a magazine assembly; 51. a magazine frame; 52. automatically lifting the sliding table; 53. a storage case; 54. a slide yoke plate; 55. a glass slide;
60. an automatic code scanning component; 61. a code scanner; 62. a code scanner fixing seat;
70. a control system component; 71. controlling an electric cabinet; 72. a multi-axis motion controller; 73. a motor driver; 74. an LED control board; 75. a 24V power supply; 76. and 5V power supply.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other examples, which a person of ordinary skill in the art would obtain without undue burden based on the embodiments of the invention, are within the scope of the invention.
In the drawings, the shape and size may be exaggerated for clarity, and the same reference numerals will be used throughout the drawings to designate the same or similar components.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The terms "first," "second," and the like in the description and in the claims, are not used for any order, quantity, or importance, but are used for distinguishing between different elements. Likewise, the terms "a," "an," or "the" and similar terms do not denote a limitation of quantity, but rather denote the presence of at least one. The word "comprising" or "comprises", and the like, is intended to mean that elements or items that are present in front of "comprising" or "comprising" are included in the word "comprising" or "comprising", and equivalents thereof, without excluding other elements or items. "upper", "lower", "left", "right", etc. are used merely to indicate relative positional relationships, which may also be changed when the absolute position of the object to be described is changed.
In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, etc. are defined with respect to the configuration shown in the drawings, and in particular, "height" corresponds to the top-to-bottom dimension, "width" corresponds to the left-to-right dimension, and "depth" corresponds to the front-to-back dimension, are relative concepts, and thus may vary accordingly depending on the location and use of the terms, and therefore these or other orientations should not be interpreted as limiting terms.
Terms (e.g., "connected" and "attached") relating to attachment, coupling, and the like refer to a relationship wherein these structures are directly or indirectly secured or attached to one another through intervening structures, as well as to a relationship wherein they are movably or rigidly attached, unless expressly stated otherwise.
In this embodiment:
as shown in fig. 1, a chromosome scanning imaging method includes the steps of:
loading a sample: placing the slide 55 smeared with the chromosome specimen on the stage assembly 30;
preliminary focusing search of a low-power objective lens: focusing the glass slide 55 in each view through the low power objective 234, and recording Z-position coordinates corresponding to the low power objective 234;
low power objective FPM reconstruction: fourier stacked microscopy imaging is performed on each field of view of the low power objective 234, a determination is made of the chromosomes within each field of view to identify metaphase chromosomes, and the metaphase chromosomes X, Y position coordinates are recorded;
high power objective lens precise focusing: according to the Z-position coordinates of the preliminary focusing of the low-power objective lens, the glass slide is precisely focused in each view through the high-power objective lens 235;
high power objective FPM reconstruction: fourier stack microscopy imaging is performed on each field of view of high power objective 235 based on metaphase chromosome X, Y position coordinates to obtain a high resolution image.
The sample loading method specifically comprises the following steps:
sequentially placing a plurality of slides 55 smeared with chromosome samples on the slide yoke plate 54;
placing a plurality of slide coupling plates 54 into the accommodation chamber of the cassette 53 from bottom to top;
driving the magazine 53 to move to a specified position;
a slide linkage 54 is loaded into a recess in the stage assembly 30 by the automatic catch assembly 40.
The preliminary focusing search of the low-power objective lens specifically comprises the following steps:
illuminating the LED flat light source 253 to illuminate the slide 55;
the control system assembly 70 drives the low power objective 234 to move along the Z direction, and obtains chromosome images through the auto-focusing assembly 22 and the camera 243;
after the imaging is clear, the focusing is stopped and the Z position coordinates of the low power objective 234 are recorded.
As shown in fig. 2, the low power objective FPM reconstruction specifically includes the following steps:
illuminating the glass slide 55 in the field of view with M LED light sources with the center LED as a symmetry center on the LED flat light sources 253 one by one at a first brightness;
the camera 243 sequentially captures M low resolution images;
initializing a low resolution image;
performing FPM reconstruction iteration;
obtaining a high-resolution image;
judging whether a metaphase chromosome exists in the high-resolution image, if so, recording X, Y position coordinates of the metaphase chromosome; if not, the low power objective lens performs preliminary focusing search on the glass slide in the next view.
The low-power objective FPM reconstruction further comprises an image denoising processing step, wherein the image denoising processing step is positioned between the steps of sequentially shooting M low-resolution images by the camera 243 and initializing the low-resolution images, and due to the existence of noise in reality, the noise is continuously accumulated and gradually influences the reconstruction result after each integral iteration; when the target information gradually becomes gentle and converges, the update duty ratio of the target information gradually decreases, and noise accumulation occupies the dominant position of the update, which eventually leads to the effect of degradation or even serious degradation of the image, so in this embodiment, after image denoising processing is performed on M low-resolution images captured by the camera 243, the FPM reconstruction iteration process is continued.
In the step of initializing the low-resolution image, the bright-field denoising image obtained by the central LED illumination is used as the amplitude and phase information of the initialized high-resolution image through up-sampling processing.
The FPM reconstruction iteration specifically comprises the following steps:
carrying out synthetic aperture operation on the low-resolution image one by one in a frequency domain by adopting an iterative method based on the combination of a synthetic aperture technology and a phase recovery algorithm;
and stopping the iterative process when the cost function value is smaller than a given threshold value by taking the cost function value as a criterion, and taking the amplitude and the phase of the high-resolution image obtained at the moment as the high-resolution image.
Fourier stacked microscopy (FPM) is a computational imaging technique that combines a synthetic aperture technique with a phase recovery algorithm. According to the technology, an LED array light source is used for replacing a traditional Kohler illumination and condenser lens system to conduct multi-angle illumination, phase information is iteratively recovered back and forth in a frequency domain by means of a phase recovery algorithm, dislocated frequency domain information is moved back to a correct position by using a Fourier translation theorem in a synthetic aperture, and the synthetic NA value of the system can reach the sum of an objective NA value and a maximum illumination NA value, so that a high-flux imaging effect with a large field of view and high resolution is achieved. At present, the FPM technology is mainly applied to pathological sections, microscope frames, drug screening and other aspects.
In this embodiment, 4 is selected × Na=0.1 objective and illumination of 0.2NA with a low power objective FPM reconstruction, resulting in a chromosome image as shown in fig. 3-b, 4 used in the present invention × The single imaging field of view of the objective lens is conventional 10 × 6.25 times the field of view of the objective lens is chosen as 10 in conventional microscope imaging as shown in FIG. 3-a × The chromosome image obtained by the NA=0.25 objective lens imaging is clearer to naked eyes, which is favorable for judging whether the chromosome image has metaphase chromosomes or not later and identifying the positions of the metaphase chromosomes for high-power reconstruction, and in addition, if 2 is used, the invention can obtain the chromosome image with the advantages of high-power reconstruction, low cost, high accuracy, and the like × The magnifying effect of the field of view can reach 25 times when the objective lens is subjected to low-power reconstruction, so that the field of view of the objective lens is larger during low-power searching, the number of fields of view of full slide searching is greatly reduced, and the scanning imaging flux of chromosome low-power searching is remarkably improved.
Judging whether metaphase chromosomes exist in the high-resolution image specifically comprises the following steps:
dividing the obtained high-resolution chromosome image into a first single independent chromosome image and an adhesion chromosome group image according to morphological characteristics of the chromosome image;
separating the adhered chromosome group image into a plurality of second single independent chromosome images by using a watershed automatic separation algorithm, a corrosion expansion algorithm and the like;
all the first single independent chromosome images and all the second single independent chromosome images are input into at least one convolutional neural network model (for example, a double-layer convolutional neural network model), chromosome class judgment is carried out, and therefore the class (including normal chromosomes and double-centromere chromosomes) to which each image belongs and the credibility probability value are obtained.
The chromosome scanning imaging method also comprises an objective lens switching step, wherein the objective lens switching step is between a low-power objective lens FPM reconstruction step and a high-power objective lens precise focusing step, and specifically comprises the following steps: the objective lens switching transmission assembly 231 drives the objective lens turntable 236 to rotate around the axis of the objective lens base 232, so that the switching action of the low-power objective lens 234 and the high-power objective lens 235 is realized.
In the high-power objective lens precise focusing step, the process also needs to complete initial focusing of a single visual field, and the focusing process uses Z-position coordinates obtained when the low-power objective lens is focused as a basic position to perform more precise automatic focusing.
As shown in fig. 4, the high power objective FPM reconstruction specifically includes the following steps:
illuminating the glass slide 55 in the field of view with N LED light sources with the center LED as a symmetry center on the LED flat light sources 253 one by one at the second brightness;
the camera 243 sequentially captures N low resolution images;
initializing a low resolution image;
performing FPM reconstruction iteration;
a high resolution image is obtained.
The high power objective 235 fourier stack microimaging reconstruction process is substantially identical to the low power objective 234 reconstruction process except that, in one aspect, the present control system assembly 70 controls the LED flat panel light source 253 to emit a second irradiance level that is higher than the first irradiance level, and N < M. On the other hand, the high-power reconstructed visual field is determined from X, Y coordinates of each metaphase chromosome recognized after the low-power reconstruction without covering the entire slide glass 55, and only the position of the metaphase chromosome is reconstructed, and the high-resolution image after the high-power reconstruction is saved as a final result of chromosome scanning imaging.
The high-resolution image reconstructed by the high-power objective FPM is shown in fig. 5-b, and compared with the high-resolution image obtained after the high-power oil lens is imaged in the conventional microscope scanning imaging method shown in fig. 5-a, the medium-term chromosome image finally obtained by the method is clearer, so that the high-resolution imaging without the oil lens is realized, and various defects caused by an automatic oil dripping system are avoided.
As shown in fig. 6, the present application further provides a chromosome scanning imaging apparatus for implementing any one of the chromosome scanning imaging methods described above, including a base 10, a microscopic imaging assembly 20, a stage assembly 30, an automatic hook assembly 40, a storage box assembly 50, and a control system assembly 70.
The base 10 is supported by four feet, and has a certain bearing strength as a supporting table surface of the whole device.
The microscopic imaging assembly 20 is the core module of the device, and as shown in fig. 7-9, the microscopic imaging assembly 20 includes a microscope frame 21, an auto-focus assembly 22, an objective lens switching assembly 23, an optical imaging assembly 24, and an LED array illumination assembly 25.
The microscope frame 21 is fixedly mounted on the base 10 as an integral support for the fully automated microscopic imaging assembly 20. In this embodiment, the microscope frame 21 is detachably connected to the base 10, so that the assembly is facilitated. In other embodiments, the microscope frame 21 and the base 10 may be integrally formed.
The automatic focusing assembly 22 comprises a focusing transmission assembly 221, a focusing transmission guide rail 222, a focusing lifting seat 223, a precise grating ruler assembly 224 and a focusing photoelectric switch 225.
The focusing transmission guide rail 222 and the focusing lifting seat 223 are both arranged on the microscope frame 21, and the focusing lifting seat 223 is in sliding connection with the focusing transmission guide rail 222.
The objective lens switching component 23 is arranged on the focusing lifting seat 223, and the focusing transmission component 221 drives the focusing lifting seat 223 to move so as to drive the objective lens switching component 23 to move along the Z direction. The focusing transmission component 221 is a motor and a precise grinding threaded screw rod, power-down self-locking can be realized while focusing stepping resolution is ensured, and the guide rail component is a high-precision crossed roller guide rail and ensures focusing guide linear precision.
The precise grating ruler component 224 and the focusing photoelectric switch 225 are both arranged on the microscope frame 21 and positioned on one side of the focusing transmission component 221 and used for carrying out closed-loop feedback on the Z-position coordinate of the focusing lifting seat 223 so as to improve the focusing position precision. Use of 40 in the present invention × The objective lens performs final reconstruction imaging with a depth of field on the order of several microns, which greatly reduces the difficulty of focus control and manufacturing of the autofocus assembly 22.
The objective lens switching unit 23 includes an objective lens switching actuator unit 231, an objective lens holder 232, an objective lens switching photoelectric switch 233, a low power objective lens 234, a high power objective lens 235, and an objective lens turret 236.
The objective lens holder 232 is mounted on the focus elevating holder 223.
The objective lens switching transmission assembly 231 is arranged on the objective lens seat 232 and is in transmission connection with the objective lens switching assembly 23, and is used for driving the whole objective lens switching assembly 23 to move along the Z direction in the focusing process. The objective lens switching transmission assembly 231 is a motor and a synchronous belt.
The objective turret 236 is mounted on the objective mount 232 and is drivingly connected to the objective switching actuator assembly 231.
The low power objective 234 and the high power objective 235 are arranged on the objective turntable 236 and positioned below the objective turntable 236, and the objective switching transmission component 231 drives the objective turntable 236 to rotate around the axis of the objective seat 232, so that the switching action of the low power objective 234 and the high power objective 235 is realized. In this embodiment, 4 is selected × Low power objective 234/na=0.1 and 40 × High power objective lens 235 with na=0.75. Use of 40 in the present invention × Objective lens feedingLine final reconstruction imaging with a field of view of 100 × Even if the stage position coordinates deviate by a factor of 6.25 times from the oil mirror by a factor of several micrometers, even by a factor of ten or more micrometers, the metaphase chromosome will still be within the field of view of the target surface of the camera 243. In the low-power searching process, the single field area is greatly improved, so that the requirement on the scanning speed of the object stage is obviously reduced.
The objective lens switch photoelectric switch 233 is disposed on the objective lens base 232 and located on one side of the objective lens switch driving component 231, and the objective lens switch photoelectric switch 233 is used as a rotation angle position feedback element and mechanically positioned in cooperation with a mechanical clamping groove on the objective lens turntable 236.
The optical imaging assembly 24 includes an imaging lens 241, a relay barrel 242, a camera 243.
An imaging lens 241 is mounted on the microscope frame 21 and above the microscope frame 21 for converging the parallel light passing through the low power objective lens 234 or the high power objective lens 235.
The transfer tube 242 has one end connected to the imaging lens 241 and the other end connected to the camera 243 for imaging an image on the focal plane of the camera 243.
The camera 243 is used to take a picture of the slide 55 and transmit the image to the control system assembly 70 for either a low power objective FPM reconstruction or a high power objective FPM reconstruction.
The LED array illumination assembly 25 includes a light source holder 251, a two-dimensional manual adjustment table 252, and an LED flat panel light source 253.
The light source holder 251 is mounted on the base 10 and is located below the stage assembly 30.
A two-dimensional manual adjustment stage 252 is mounted on the light source holder 251 for manual fine adjustment of the position of the LED flat panel light source 253.
The LED flat panel light sources 253 are movably mounted on the upper surface of the two-dimensional manual adjustment table 252 and are communicatively connected to the control system assembly 70, and the control system assembly 70 adjusts the light emission brightness and the light emission quantity of the LED flat panel light sources 253 according to the low power objective 234 or the high power objective 235. As the illumination source of the microscopic imaging assembly 20, the LED flat light source 253 should be noted to ensure that the LED position in the center of the LED flat light source 253 and the target surface of the camera 243 before the system starts to operateIf the centers are not overlapped, the two-dimensional manual adjustment table 252 is adjusted to be overlapped and then locked. In this embodiment, the number of the LED flat light sources 253 is 2 and can be automatically switched, which is different from the components such as the light source, the condenser, the electric diaphragm, etc. in the conventional full-automatic microscope, and the low power and high power objective lenses are respectively adapted to perform FPM imaging, and the automatically switched low power (2 × Na=0.08 or 4 × Na=0.1) and a high power objective lens (40 × Na=0.75 or 60 × Na=0.9) are used for searching and final imaging, respectively, to achieve large field of view high flux searching imaging of chromosomes and oil-free mirror final imaging. The LED flat light source 253 has a maximum illumination NA of 0.5, and can be used with 40 when high-power illumination is performed × Na=0.75 dry objective lens to synthesize an equivalent NA value of 1.25, compared to conventional 100 × Na=1.25 oil mirror equivalents; in low-power illumination, the brightness of LEDs needs to be reduced, and more dense LED light sources are lightened, and the illumination and reconstruction speed are considered, so that the maximum NA illumination is not adopted, and only the central part of an LED array is used for sequentially illuminating, so that the illumination NA of 0.2 can be realized, and the illumination NA can be matched with 4 × Objective lens with na=0.1 synthesizes equivalent NA value of 0.30, compared with the traditional 10 × Na=0.25 objective lens.
As shown in fig. 11, the stage assembly 30 includes an X-direction transmission assembly 31, a Y-direction transmission assembly 32, a stage lower plate 33, a stage middle plate 34, a stage upper plate 35, an X-direction guide assembly 36, a Y-direction guide assembly 37, and a scanning stage photoelectric switch 38.
The stage lower plate 33 is fixedly mounted on the microscope frame 21 as a support for other components in the stage assembly 30.
The stage intermediate plate 34 is mounted on the stage lower plate 33 via a Y-direction guide rail assembly 37, and scanning movement of the stage intermediate plate 34 in the Y-direction is achieved via a Y-direction transmission assembly 31.
The stage upper plate 35 is mounted on the stage middle plate 34 via an X-direction guide assembly 36, and scanning movement of the stage upper plate 35 in the X-direction is achieved via the X-direction transmission assembly 32. In this embodiment, the X-direction transmission assembly 31 and the Y-direction transmission assembly 32 are motors and precision ball screws, and the rail assemblies are linear rail sliders.
The scanning table photoelectric switch 38 is arranged on the object stage middle plate 34, and the scanning table photoelectric switch 38 is used as a position zero point and an electric limit for improving the positioning accuracy of the scanning table and avoiding the overrun of the travel.
As shown in fig. 10, the automatic hook sheet assembly 40 includes a hook sheet driving assembly 41, a hook sheet guide assembly 42, a hook sheet metal 43, and a T-shaped hook 44.
The hook rail assembly 42 is mounted on the stage upper plate 35.
The hook sheet metal 43 is fixedly arranged on the sliding block of the hook sheet guide rail assembly 42, the hook sheet metal 43 is driven to move along the X direction by the hook sheet transmission assembly 41, and the hook sheet transmission assembly 41 is a motor and a synchronous belt, so that rapid loading and unloading actions of the slide yoke plate 54 can be realized.
The T-shaped hooks 44 are fixedly mounted on the hook sheet metal 43, and the dimensions of the T-shaped hooks are matched with those of the T-shaped grooves on the slide yoke plate 54, so that the slide yoke plate 54 can be automatically loaded into the grooves on the upper plate 35 of the object stage, and after scanning imaging is completed, the slide yoke plate 54 is unloaded into the storage box 53.
As shown in fig. 12, the magazine assembly 50 includes a magazine frame 51, an automatic elevation slide table 52, a magazine 53, a slide yoke 54, and slides 55.
The magazine frame 51 is mounted on the support base 10 as a support structure for other components in the magazine assembly 50.
The automatic lifting sliding table 52 is mounted on the magazine frame 51, and the extending direction of the automatic lifting sliding table 52 is perpendicular to the plane of the base 10.
The storage box 53 is slidably mounted on a table top of the automatic lifting sliding table 52, and can perform a Z-direction lifting motion, and a containing cavity is arranged inside the storage box 53 and used for placing a plurality of slides 55 smeared with chromosomes. In this embodiment, 30 slide connecting plates 54 are installed from the inside of the storage box 53 to the bottom and up, 6 slides 55 are installed in each slide connecting plate 54, the number of the maximum single specimens in the whole storage box 53 is 180, the device continuously scans 6 slides 55 by hooking the specimens at a single time, and continuously and automatically scans 180 specimens at a single startup, so as to realize the requirement of automatic scanning without unattended operation at night. The elevation travel of the magazine 53 matches the height of the table top of the stage assembly 30, ensuring that all 30 slide linkage plates 54 can be hooked by the auto hook assembly 40 and moved into the table top of the stage assembly 30 for scanning imaging by the change in height.
The automatic code scanner 60 includes a code scanner 61 and a code scanner holder 62.
The scanner mount 62 is fixedly mounted to the microscope frame 21 and is positioned between the fully automated microscopic imaging assembly 20 and the magazine assembly 50.
The code scanner 61 is arranged on the code scanner fixing seat 62 and is used for reading and storing two-dimensional code information of a patient on a sample slide in the process of hooking the sample slide.
The control system assembly 70 is independently positioned to provide automated control functions for the overall device, as shown in fig. 13, and includes a control electronics cabinet 71, a multi-axis motion controller 72, a motor drive 73, and an LED control board 74.
The control cabinet 71 is used for storing the individual control, drive and power supply units in the device.
The multi-axis motion controller 72 is used to control the motion of the 6 motors in the system.
The motor driver 73 is used to drive 6 motors in the device.
The LED control board 74 is used to control the lighting timing and brightness adjustment of the LEDs in the LED panel light source 253.
A 24V power supply 75 is used to power the multi-axis motion controller 72.
The 5V power supply 76 is used to power the LED control board 74. As shown in fig. 14, the control system assembly 70 sends instructions through the upper computer software, and performs signal transmission with the multi-axis motion controller 72, the LED control board 74 and the camera 243 through the internet access, the serial port and the USB, respectively.
In the application, the microscopic imaging component 20 and the storage box component 50 are installed on the left side and the right side of the base 10 in parallel, and the automatic hooking component 40 is installed in the objective table component 30 in an embedded manner, so that full-automatic, rapid and high-flux scanning imaging is realized. The chromosome scanning imaging device has compact arrangement of all modules, small occupied space, small manufacturing difficulty and low production cost. The invention adopts the Fourier microscopic laminated imaging technology to scan and image the chromosome, in the process of low-power searching, the field of view of the objective lens is larger, the number of fields of view of the whole glass slide searching is greatly reduced, and the scanning and imaging flux of the chromosome low-power searching is obviously improved; in the high-power final imaging process, the high-resolution imaging without the oil mirror is realized, and various defects caused by an automatic oil dripping system are avoided; the invention greatly reduces the requirements of positioning precision, running speed and feeding resolution of a plurality of core modules of the automatic chromosome scanning imaging device, and can greatly reduce the development cost and difficulty of the system.
The invention also provides a detailed using method of the chromosome scanning device, the concrete working flow is shown in figure 15, the storage box 53 filled with the sample slide glass 55 is put in place, and the automatic slide scanning flow is started; the control system assembly 70 controls the stage assembly 30 to move to a preset position and controls the automatic hook piece assembly 40 to extend the T-hook 44 to a designated position; the control system assembly 70 controls the elevation movement of the storage box assembly 50 to a designated position, the T-shaped hooks 44 hook the T-shaped grooves on the slide yoke plate 54 and load the T-shaped grooves into the grooves on the objective table upper plate 35, and meanwhile, in the process of hooking, the code scanner 61 scans the two-dimensional codes on each sample slide 55 to register patient information; the control system assembly 70 controls the stage assembly 30 to move to the initial position for the scan, in preparation for starting the scan process; after the scanning and algorithmic reconstruction of one slide 55 are completed by the fourier stack microscopy (FPM) technique, the same operation flow of the next slide 55 is continued, and the control system assembly 70 controls the stage assembly 30 to move to a preset position of the next slide 55, and the scanning and reconstruction processes are repeated until the scanning and imaging of 6 slides 55 in one slide link 54 are completed, and the control system assembly 70 controls the automatic catch assembly 40 to unload the slide link 54 into the storage bin 53; next, the control system assembly 70 controls the elevation movement of the storage box assembly 50 to a designated position, the T-shaped hooks 44 hook the T-shaped grooves on the next slide yoke plate 54, load them into the grooves on the stage upper plate 35, and repeat the scanning, reconstructing and unloading processes until the chromosome images reconstructed from all 180 slides 55 in the 30 slide yoke plates 54 are stored, completing the whole process of automated high-throughput chromosome oil-free mirror scanning imaging.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art. Although embodiments of the invention have been disclosed above, they are not limited to the use listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. Therefore, the invention is not to be limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (10)
1. A chromosome scanning imaging method, characterized in that: the method comprises the following steps:
loading a sample: placing the slide with the chromosome sample smeared thereon on the stage assembly;
preliminary focusing search of a low-power objective lens: focusing the glass slide in each view through the low-power objective lens, and recording Z-position coordinates corresponding to the low-power objective lens;
low power objective FPM reconstruction:
illuminating M LED light sources which take a central LED as a symmetrical center on the LED flat light sources one by one with first brightness to illuminate the glass slide in each view;
the camera shoots M low-resolution images in sequence;
initializing a low resolution image;
performing FPM reconstruction iteration;
obtaining a high-resolution image;
judging whether a metaphase chromosome exists in the high-resolution image, if so, recording X, Y position coordinates of the metaphase chromosome; if not, repeating the FPM reconstruction step of the low power objective lens in the next field of view;
high power objective lens precise focusing: according to the Z-position coordinates of the preliminary focusing of the low-power objective lens, carrying out precise focusing on the glass slide in each view through the high-power objective lens;
high power objective FPM reconstruction:
illuminating the glass slides in each view by illuminating N LED light sources on the LED flat light sources one by one with second brightness and taking the central LED as a symmetrical center according to the position coordinates of the metaphase chromosome X, Y;
the camera shoots N low-resolution images in sequence;
initializing a low resolution image;
performing FPM reconstruction iteration;
obtaining a high-resolution image;
the second luminance is higher than the first luminance, the N < M.
2. The chromosome scanning imaging method of claim 1, wherein: the FPM reconstruction iteration specifically comprises the following steps:
carrying out synthetic aperture operation on the low-resolution image one by one in a frequency domain by adopting an iterative method based on the combination of a synthetic aperture technology and a phase recovery algorithm;
and stopping the iterative process when the cost function value is smaller than a given threshold value by taking the cost function value as a criterion, and taking the amplitude and the phase of the high-resolution image obtained at the moment as the high-resolution image.
3. The chromosome scanning imaging method of claim 1, wherein: the low-power objective FPM reconstruction further comprises an image denoising processing step, wherein the image denoising processing step is positioned between the steps of shooting M low-resolution images sequentially by the camera and initializing the low-resolution images.
4. The chromosome scanning imaging method of claim 1, wherein: the preliminary focusing search of the low-power objective lens specifically comprises the following steps:
illuminating an LED flat light source to illuminate the slide;
the control system component drives the low-power objective lens to move along the Z direction, and chromosome images are obtained through the automatic focusing component and the camera;
after the imaging is clear, focusing is stopped, and Z-position coordinates of the low-power objective lens are recorded.
5. The chromosome scanning imaging method of claim 1, wherein: the loading sample specifically comprises the following steps:
sequentially placing a plurality of slides smeared with chromosome samples on a slide yoke plate;
placing a plurality of slide yoke plates in a containing cavity of a storage box from bottom to top;
driving the storage box to move to a designated position;
a slide linkage is loaded into the recess in the stage assembly by the automatic catch assembly.
6. The chromosome scanning imaging method of claim 1, wherein: the method further comprises an objective switching step, wherein the objective switching step is between the low power objective FPM reconstruction step and the high power objective precise focusing step, and specifically comprises the following steps: the objective turntable is driven to rotate around the axis of the objective seat by the objective switching transmission component, so that the switching action of the low-power objective and the high-power objective is realized.
7. A chromosome scanning imaging apparatus, characterized in that: the chromosome scanning imaging device is used for implementing the chromosome scanning imaging method according to any one of claims 1-6, and comprises a base, a microscopic imaging component, a stage component and a control system component, wherein the stage component is movably arranged on the base, a glass slide is placed on the stage component, the microscopic imaging component comprises an objective lens switching component and an LED array illumination component, the objective lens switching component is movably arranged on the base, the objective lens switching component comprises a low-power objective lens and a high-power objective lens, the low-power objective lens is used for carrying out preliminary focusing on a glass slide in a single view, the control system component controls the high-power objective lens to carry out precise focusing on each view according to Z-position coordinates of the preliminary focusing of the low-power objective lens, the control system component recognizes and positions a middle-stage chromosome in each view after the Fourier lamination imaging of the low-power objective lens, and the high-power objective lens carries out Fourier lamination microscopic imaging according to the position coordinates of the middle-stage chromosome X, Y so as to acquire high-resolution images; the LED array lighting assembly comprises an LED flat light source, the LED flat light source is in communication connection with the control system assembly, and the control system assembly adjusts the luminous brightness and the luminous quantity of the LED flat light source according to the low-power objective lens or the high-power objective lens.
8. The chromosome scanning imaging apparatus of claim 7, wherein: the LED array lighting assembly further comprises a light source support and a two-dimensional manual adjustment table, the light source support is arranged on the base, the two-dimensional manual adjustment table is arranged on the light source support and used for manually fine-adjusting the position of the light source, and the LED flat light source is movably arranged on the table top of the two-dimensional manual adjustment table and is in communication connection with the control system assembly.
9. The chromosome scanning imaging apparatus of claim 7, wherein: the object stage assembly comprises an X-direction transmission assembly, a Y-direction transmission assembly, an object stage lower plate, an object stage middle plate and an object stage upper plate, wherein the object stage lower plate is fixedly arranged on the base, the object stage middle plate is movably arranged on the object stage lower plate, and the Y-direction transmission assembly drives the object stage middle plate to perform scanning motion along the Y direction; the object stage upper plate is movably arranged on the object stage middle plate, and the X-direction transmission component drives the object stage upper plate to perform scanning motion along the X direction.
10. The chromosome scanning imaging apparatus of claim 7, wherein: the microscopic imaging assembly further comprises a microscope frame and an automatic focusing assembly, the microscope frame is arranged on the base, the automatic focusing assembly comprises a focusing transmission assembly and a focusing lifting seat, the focusing lifting seat is arranged on the microscope frame and is in transmission connection with the focusing transmission assembly, the objective lens switching assembly is arranged on the focusing lifting seat, and the focusing transmission assembly drives the focusing lifting seat to move so as to drive the low-power objective lens or the high-power objective lens to focus along Z-direction movement.
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