CN112114423A - Portable full-automatic multi-mode microscopic imaging device - Google Patents
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Abstract
The invention discloses a portable full-automatic multi-mode microscopic imaging device, which comprises a two-dimensional electronic control scanning platform, an automatic focusing system and a multi-mode microscopic imaging device based on a color coding LED array. In the multimode imaging, an LED array with color coding is used as a light source of a microscope system to generate color illumination of different modes, so that three imaging modes of bright field imaging, dark field imaging and differential phase-contrast imaging are realized. The complete imaging mode does not need to dye and fluorescently mark cells, and the growth process and the growth state of living cells can be observed for a long time by combining the automatic maintenance of a stable culture environment in the device.
Description
Technical Field
The invention relates to a portable full-automatic multi-mode optical imaging system which is convenient for field scientific research and disease diagnosis of rural clinics.
Background
The optical microscope is an optical instrument for conveniently obtaining the structural information of the fine substance by people, and has important application in the fields of medicine, biology, material analysis, electronic element detection and the like. Over the past decades, with the continued sophistication of microscopic imaging techniques, many new imaging modalities have emerged. Bright field imaging, dark field imaging and differential phase contrast microscopy are still the three most widely used imaging modes today.
Bright field microscopy images are acquired based on the difference in light absorption at different parts of the sample. Therefore, for transparent or translucent phase-only samples, such as unlabeled cells and thin tissue specimens, the imaging effect obtained is not ideal because of the difficulty in modulating the transmitted light intensity.
The chemical dyeing and fluorescent labeling method dyes the sample by utilizing different affinities of different components in the cell to different chemical substances or fluorescent dyes to form light intensity contrast or generate different spectrums, thereby realizing the labeling imaging of the transparent sample. Both the traditional dye staining method and the biological protein marker luminescence method inevitably have certain influence on the sample due to exogenous substances. In particular, in the long-term observation of living cells, the cells are easily damaged by excitation light, and the fluorescent group has a photobleaching problem.
Dark field microscopy imaging techniques are the opposite of bright field microscopy imaging techniques. When the sample is viewed under a dark field microscope, the observer sees the sample appearing bright on a dark background. Dark field microscopy uses oblique illumination at angles exceeding the maximum angle that can be captured by the numerical aperture of the optical imaging system, and collects scattered light to obtain high frequency detail information of the sample.
The method can generate high-contrast images, is sensitive to the edges of the samples, and has certain advantages for imaging the samples with weak absorption and clear outlines. Researchers typically observe surface details of a sample using dark field microscopy. However, there is also a case where the imaging effect is poor due to the overall image being dark due to weak signal light.
For the amplitude sample, because the transmittance distribution is different when light penetrates through the sample, the amplitude intensity changes, and bright field imaging and dark field imaging can acquire different types of sample information. For a transparent, semitransparent and especially pure phase sample, there is almost no change in amplitude when light passes through the sample, and then bright field imaging and dark field imaging cannot image the appearance of the sample, so that the two imaging modes are limited in that only information about the change in amplitude of the light transmitted by the sample can be obtained.
How to clearly observe transparent samples such as biological slices, phase gratings and the like without a destructive staining process. The advent of phase contrast microscopy imaging technology is said to provide a good solution to the above-mentioned problems. The phase change of light passing through a transparent sample is converted into amplitude change by a condenser lens with an annular diaphragm and a phase difference objective lens with a phase plate, so that a three-dimensional pseudo-stereoscopic image of the sample can be obtained, and the three-dimensional pseudo-stereoscopic image has strong stereoscopic impression and can be directly observed by human eyes and a camera.
Similar to the phase contrast microscopy imaging technology, the differential phase contrast microscopy imaging mode based on the LED array is also a common way to obtain a transparent sample picture, and the method is to perform time-sharing imaging on illumination light generated by the LED array in different areas, and perform differential imaging on the two images. Differential phase contrast imaging mode based on LED arrays can produce phase contrast microscopic imaging mode effects.
Whether a transparent sample or a non-transparent sample is subjected to imaging, the three imaging modes of bright field imaging, dark field imaging and differential phase contrast can be combined to completely provide information of the sample. At the moment, the above three imaging modes all need different hardware supports, including condenser diaphragms, polarizing elements or special optical devices. This invisibly increases the complexity of the optical path adjustment and does not allow the simultaneous acquisition of three types of imaging information during the imaging process.
In recent years, with the development of optoelectronics technology, LED illumination has been intensively studied as a light source for microscope imaging. The color coding LED array is introduced into microscopic imaging, bright field, dark field and differential phase contrast imaging can be realized simultaneously, and the problem of long-time observation of unmarked living cells is well solved.
In addition, for long-time observation of a sample, the mechanical stress of the microscope inevitably causes imaging focal plane offset, and manual focusing not only increases the complexity of an experiment, but also influences the experiment precision due to visual deviation. On the other hand, multi-region sample observation requires displacement of the sample platform. Therefore, the portable multi-mode microscopic imaging device has positive significance for rural diagnosis and treatment.
Disclosure of Invention
The invention aims to provide a portable full-automatic multi-mode microscopic imaging device. Through the miniaturized microscope hardware structure of optoelectromechanical design, adopt the illumination of colored coding to form multiple imaging mode, improve the imaging quality, reach imaging mode diversified. The device with miniaturized instrument scale and automatic control system is designed.
The invention provides a full-automatic multi-mode microscopic imaging device, which comprises an optical imaging system shell, a color coding LED matrix, a sample moving platform, an objective lens, a reflector, a lens barrel, an imaging lens, a camera, a one-dimensional axial displacement platform for driving the objective lens to move up and down, wherein the color coding LED matrix is arranged above the sample moving platform, light rays are emitted from the color coding LED matrix, pass through the sample moving platform, a sample enters the lens barrel through the reflector after being amplified by the objective lens, the imaging lens is arranged in the lens barrel, the sample is imaged on the camera, the device also comprises a main control unit for controlling the objective lens to carry out axial scanning around an imaging area in the sample imaging process, an image is acquired by the camera, the optimal imaging axial position is obtained through a definition imaging algorithm, the objective lens is driven to the imaging clear position, and an illumination pattern of the color coding LED matrix is configured, clear images collected by a camera are processed by channels, and bright field imaging, dark field imaging and differential phase contrast microscopic imaging are obtained at one time.
The invention has the following advantages and effects:
(1) and the multimode imaging can meet the requirements of various researches on sample observation. Multimodal imaging helps to achieve a more comprehensive viewing experience.
(2) The full-automatic microscope function reduces experimental error. The auto-focus function reduces visual offset of manual focusing in the experiment. The two-dimensional electronic control scanning platform can realize multi-view field observation of the sample.
In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the drawings.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
fig. 1 is an overall architecture diagram of a fully automated multi-modality microscopic imaging apparatus according to the present invention.
Wherein, 1, an optical imaging system shell; 2. a circular color LED matrix; 3. a sample moving platform (sample stage); 4. an objective lens; 5. a mirror; 6. a lens barrel; 7. an imaging lens; 8. a camera; 9. a sample stage scanning motor; 10. a one-dimensional axial displacement stage; 11. a diaphragm plate; 12. lifting the cantilever; 15. lens cone fixing seat.
Fig. 2 is a schematic diagram of the internal structure of the fully automatic multi-modality microscopic imaging apparatus according to the present invention.
Fig. 3 is an exploded view of a fully automated multi-modality microscopic imaging apparatus according to the present invention.
Fig. 4 is a control flow diagram of the fully automatic multi-modality microscopic imaging apparatus according to the present invention.
Fig. 5 is a multi-mode imaging optical path diagram according to the present invention.
Wherein, 1, an imaging system; 2. color coded LED illumination; 3. an experimental sample stage; 4. an objective lens; 5. a tube lens; 6. a CCD camera; 7. an experimental sample; 8. red illumination light; 9. a blue illumination light; 10. and green illumination light.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
Fig. 1-5 illustrate some embodiments according to the invention.
The invention designs miniaturized microscope hardware by optoelectromechanical, and integrates control and imaging algorithms of system hardware by self-programming control software, thereby achieving full-automatic focusing and scanning multi-mode microscopic imaging. The temperature and carbon dioxide concentration regulation function is realized in the device.
The miniature microscope hardware system is shown in fig. 1-3, and comprises an optical imaging system shell 1, a circular color coding LED matrix 2, a sample moving platform 3, an objective 4, a reflector 5, a lens cone 6, an imaging lens 7, a camera 8, a sample stage scanning motor 9, a one-dimensional axial displacement stage 10, a diaphragm plate 11 and a lifting cantilever 12.
The optical imaging system shell 1 is a box body, a transverse partition plate is arranged in the optical imaging system shell 1, the sample moving platform 3 is arranged on the transverse partition plate, the one-dimensional axial displacement platform 10 is arranged close to the left side wall of the shell 1 and is provided with a lifting cantilever 12 located below the transverse partition plate, an objective lens 4 is supported on the lifting cantilever 12, the reflector 5 is located below the lifting cantilever 12 and is arranged at 45 degrees, the lens cone 6 is horizontally and transversely arranged and is installed on a shell bottom plate through a lens cone fixing seat 15, and the camera 8 is located on the outer side of the shell 1.
The software control flow is shown in fig. 4. The master control computer controls the whole device to realize full automation, including motion control, LED illumination control and camera acquisition. The X axis and the Y axis are controlled by movement to realize sample scanning or field conversion, and the Z axis realizes the automatic focusing function. The acquired images are simply calculated to obtain bright field, dark field and differential phase contrast imaging.
The project is developed from the aspects of opto-electro-mechanical design, mechanical manufacturing, software control, imaging algorithm, microscopic imaging verification and the like.
The optical electromechanical design comprises three parts of a microscopic imaging light path, illumination control and an electric control platform. And (3) constructing a basic structure according to a microscope imaging system by adopting an infinite microscopic imaging scheme and a fixed imaging lens and camera distance. A one-dimensional miniaturized electric control displacement platform is built by adopting a micro motor, the axial motion of an objective lens is controlled, and the relative control of the distance between a sample and the objective lens is realized. The LED array is controlled to illuminate through the single chip microcomputer, and subsequent self-programming control software is matched, so that the illumination structure is reduced, and diversified illumination is freely controlled. A miniature two-dimensional microscopic platform driven by a micro motor is adopted to be matched with various sample pools, so that two-dimensional control movement of samples is achieved.
In mechanical manufacturing, the structure of the electric control platform, various fixing pieces/adapters and the whole machine shell are designed through three-dimensional modeling. Adopt 3D to print the part that has the complex construction inside the preparation by oneself, the processing preparation is easily processed the part. The whole structure of the instrument is realized by purchasing various parts for assembly.
In software control, LabVIEW trigger control signals are adopted to realize the control of the state of the single chip microcomputer, so that bright field, dark field and differential illumination of the LED array are obtained. In the aspect of camera acquisition imaging, an image acquisition module is self-compiled in LabVIEW, and the functions of real-time imaging and basic acquisition (storage, exposure time, ROI and the like) of a microscopic image are realized.
In the aspect of motor control, LabVIEW is used for driving the motor to move through the motion control card, and a limit signal is fed back from a motion position to realize specific motion, including linear acceleration motion and deceleration motion. The two-dimensional scanning of the sample and the one-dimensional axial scanning of the objective lens are realized by combining the driving of a multi-axis motor. And on a software interface designed by LabVIEW, the control of illumination, imaging and a motor is realized in a label mode, and corresponding parameters are locked during switching.
The imaging system constructed by the invention can realize three modes of bright field, dark field and differential phase contrast. A color coding LED circular array is used as a light source of an imaging system, and light from the light source is directly projected into an objective lens in a bright field imaging mode. The dark field imaging mode refers to the light source using the colored LED illumination mode above fig. 5 to implement a single lens multi-mode imaging system. The illumination pattern consists of two semicircles, a Red (Red) semicircle 8 and a Blue (Blue) semicircle 9, and a Green (Green) outer circle region 10.
R, B the radius of the semi-circle is determined by the numerical aperture of the imaging system. Thus, the bright field illumination area is set to red and blue, the green LED is dark field illumination, and the corresponding illumination angle is greater than the numerical aperture maximum of the imaging system. The color illumination pattern is used to achieve single lens multi-contrast imaging.
The numerical aperture formula of the objective lens shows that:
NA0＝n·sinθ--------------------(1)
wherein n is a medium through which light passes; theta is the maximum angle of incidence at which the objective ray can enter.
From FIG. 5:
from equations (1), (2), the maximum radius R of the color-codable LED circular array that can enter the objective lens is given by:
when the objective lens NA0When determining, let n be 1, so the bright field illumination range radius R of the color-encodable LED circular array is determined, i.e., the two semi-circle radii of Red (Red) and Blue (Blue) are determined. The remaining part of the LEDs is defined for dark field illumination, i.e. the outer circle region of Green (Green).
Coding and illuminating according to the value of the illumination radius R, and performing three-channel separation on the image obtained by the color CCD to obtain a red channel image (I)Red) Blue channel image (I)Blue) And green channel image (I)Green)。
Bright field image IBMExpressed as:
IBM＝IRed+IBlue----------------------------------(4)
dark field image IDMExpressed as:
IDM＝IGreen----------------------------------(5)
differential phase contrast image IDPCExpressed as:
the camera can acquire one image and realize three imaging modes simultaneously.
The bright field illumination imaging mode can collect low-frequency information of the sample and is suitable for observing the sample with strong absorption. The dark field microscope can acquire high-frequency information of the sample and is suitable for observing detail change of the sample. The differential phase contrast microscope is suitable for samples with weak absorption, and image information can be obtained for transparent samples without dyeing.
In the invention, an objective lens carries out axial scanning around an imaging area, a camera (8) collects a group of images with axial position marks, a definition evaluation value F of each image is calculated through a definition imaging algorithm, and the position of the image corresponding to the maximum value of the definition evaluation value F is taken as the optimal imaging axial position.
The autofocus control of the present invention uses the Brenner gradient function of the improved algorithm as the focus sharpness evaluation function. The improved Brenner gradient function judges the imaging quality, records the corresponding axial position, improves the algorithm that the Brenner gradient function changes more stably when the best imaging plane is obtained and the feedback motor moves to the clear imaging position, can effectively remove noise interference and can quickly finish automatic focusing.
Assuming that the gray value of the point (x, y) in the image of size M N is I (x, y), the Brenner gradient function of the improved algorithm is given by the formula:
the invention relates to a self-designed miniaturized two-dimensional microscopic electric control platform which is driven by a micro motor and is adaptive to various sample pools, thereby achieving two-dimensional control motion of samples.
The sample moving platform can move in two dimensions through the stepping motor. The electric control two-dimensional platform is not limited to the design structure shown in fig. 1, and may be other structures, such as a manual displacement table, a splicing two-dimensional electric control translation table, and a square connected two-dimensional electric control translation table.
The imaging device is not limited to the inverted transmission type microscopic imaging mode, and the design mode is also suitable for the upright and reflection type imaging devices.
The invention utilizes color coding illumination to calculate the obtained single-frame image channel after separation, thereby realizing three imaging modes. The color-coded illumination device is not limited to the shape shown in fig. 1, and is also applicable to rectangular illumination as long as color coding can be performed.
In the present invention, the housing 1 serves as an incubator, in which carbon dioxide gas is filled, and the main control unit also serves to control the concentration and temperature of the carbon dioxide gas. In the prior art, the incubator is temporarily placed on a sample object stage during microscopic imaging, is inconvenient to use and carry due to combination use, and the microscopic imaging device is portable and easy to use.
In the invention, the complete imaging mode does not need to dye and fluorescently label the cells, and the growth process and the growth state of the living cells can be observed for a long time by combining the automatic maintenance of a stable culture environment in the device.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A full-automatic multi-mode microscopic imaging device comprises an optical imaging system shell (1), a color coding LED matrix (2), a sample moving platform (3), an objective lens (4), a reflector (5), a lens cone (6), an imaging lens (7), a camera (8) and a one-dimensional axial displacement platform (10) for driving the objective lens (4) to move up and down,
the color coding LED matrix (2) is arranged above the sample moving platform (3), light rays are emitted from the color coding LED matrix (2) and pass through the sample moving platform (3), a sample is amplified by the objective lens (4) and then enters the lens barrel (6) through the reflector (5), the imaging lens (7) is arranged in the lens barrel (6) to image the sample on the camera (8),
the system also comprises a main control unit, wherein the main control unit is used for controlling the objective lens to carry out axial scanning around an imaging area in the sample imaging process, acquiring images by the camera (8) and obtaining the optimal imaging axial position through a definition imaging algorithm, driving the objective lens (4) to the clear imaging position, acquiring clear images by the camera (8) through channel division processing by configuring an illumination pattern of the color coding LED matrix (2), and obtaining bright field imaging, dark field imaging and differential phase contrast microscopic imaging at one time.
2. The fully automated multi-modality microscopic imaging apparatus according to claim 1, wherein the sample moving stage (3) is a two-dimensional electrically controlled translation stage for two-dimensional scanning of a sample during imaging of the sample.
3. The fully automated multi-modality microscopic imaging apparatus according to claim 1, wherein the color-coded LED matrix (2) is disposed above a sample stage with a center LED bead disposed at an imaging system optical axis position.
4. The fully automated multimodal microscopic imaging apparatus according to claim 1, wherein the color coded LED matrix (2) is a circular or rectangular part.
5. The fully automatic multi-modal microscopic imaging apparatus according to claim 1, wherein the illumination pattern is composed of two semicircles of a red semicircle and a blue semicircle and a green outer circle region, red and blue are set in the bright field illumination region, the green outer circle region is dark field illumination, and the corresponding illumination angle is larger than the maximum value of the imaging system.
6. The fully automatic multi-modal microscopic imaging apparatus according to claim 5, wherein three channels of images obtained by the camera are separated to obtain a red channel image IRedBlue channel image IBlueAnd green channel image IGreenWhereas from the three-channel image a bright-field image I is obtainedBMDark field image IDMDifferential phase contrast image IDPC。
7. The fully automated multi-modality microscopic imaging apparatus according to claim 1, wherein the imaging layout is inverted transmission microscopic imaging.
8. The fully automated multi-modality microscopic imaging apparatus according to claim 1, wherein the objective magnification is replaced by an RMS style objective screw interface.
9. The fully automated multi-modality microscopic imaging apparatus according to claim 1, wherein a diaphragm is provided within the optical imaging system housing (1), the sample moving stage (3) is provided on the diaphragm, and the one-dimensional axial displacement stage (10) is disposed adjacent to a housing sidewall, having a lifting cantilever located below the diaphragm, wherein,
the objective lens is supported on the lifting cantilever, the reflector (5) is positioned below the lifting cantilever and arranged at an angle of 45 degrees, the lens barrel (6) is horizontally and transversely arranged, and the camera (8) is positioned outside the shell (1).
10. The fully automated multi-modality microscopic imaging apparatus according to any one of claims 1 to 9, wherein the optical imaging system housing (1) serves as an incubator filled with carbon dioxide gas, and the main control unit is further used for controlling the concentration and temperature of the carbon dioxide gas.
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| CN114088606A (en) * | 2021-10-23 | 2022-02-25 | 广州市艾贝泰生物科技有限公司 | Cell analyzer |
| CN115047614A (en) * | 2022-05-24 | 2022-09-13 | 吉林大学 | Microscopic imaging automatic detection method and system based on full-automatic displacement table |
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