CN219737242U - Phytoplankton bright field and fluorescence dual-light path synchronous imaging device - Google Patents
Phytoplankton bright field and fluorescence dual-light path synchronous imaging device Download PDFInfo
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Abstract
The utility model relates to a phytoplankton bright field and fluorescence dual-light path synchronous imaging device, which comprises: the double-light-path imaging optical system is respectively connected to the light source control circuit and the upper computer control system; the double-light-path imaging optical system comprises a bright-field imaging light path and a fluorescent imaging light path, and the two light paths share a microscopic imaging objective lens and a beam splitting prism; the light splitting prism is used as a center, the bright field imaging light path adopts transmission type illumination, the fluorescent imaging light path adopts falling type illumination, the light splitting mode after the microscope objective is adopted, and the imaging vision field of the bright field imaging light path is consistent with that of the fluorescent imaging light path; after the light is split by the microscope objective, the bright field imaging light path and the fluorescent imaging light path are in symmetrical structures by the symmetrical planes of the splitting surfaces of the splitting prism, and the light paths of the two light paths after light splitting reach the respective cameras are consistent.
Description
Technical Field
The utility model belongs to the field of resources and environment, in particular to a phytoplankton bright field and fluorescence dual-light path synchronous imaging device.
Background
The method is significant in the aspects of identifying and counting the types and the number of phytoplankton, judging the eutrophication degree in water areas, early warning water bloom and the like. The current classification and identification method of phytoplankton mainly comprises an identification method based on morphological characteristics of phytoplankton and an identification method based on fluorescence spectra of phytoplankton fluorescence characteristics. The morphological detection method is also called microscopic imaging method, has the advantages of visual recognition process, high recognition precision and the like, but has the problems that phytoplankton cells with similar forms are difficult to distinguish, the recognition process is influenced by impurity in water, the image resolution and the like.
The phytoplankton fluorescent image contains rich phytoplankton autofluorescence pigment information, and the phytoplankton fluorescent image assisted microscopic image registration recognition technology is utilized, so that the interference of water impurities can be effectively eliminated, the accurate positioning and contour segmentation of the phytoplankton cell position can be effectively realized, the phytoplankton pigment types can be distinguished, and the phytoplankton identification accuracy can be improved. The Walker et al performs gray level and fluorescence imaging positioning matching on phytoplankton cells, performs classification and identification of anabaena and microcystis through an algorithm, has the classification accuracy of more than 97%, and can be effectively applied to complex water sample identification. Burkhard et al demonstrate significant advantages for distinguishing phytoplankton from impurities, phycocyanin-containing phytoplankton and non-phycocyanin phytoplankton using a phytoplankton bright field fluorescent image by integrating the phytoplankton bright field fluorescent microscopic imaging information.
At present, a single-light-path inverted fluorescence microscope imaging system is mainly adopted for acquiring the bright field-fluorescence microscope image of the phytoplankton, the bright field and fluorescence imaging mode switching is realized through manual operation, the switching process is influenced by factors such as water body flow, living phytoplankton cells self-swimming and the like, random dislocation exists between the bright field image and the fluorescence image of the phytoplankton cells under microscopic vision, and the registration of the bright field and the fluorescence image faces a great challenge. In order to acquire the bright field and fluorescence microscopic images of the phytoplankton cells corresponding to registration, bi and the like acquire the hyperspectral images of the phytoplankton by combining a single-camera with a liquid crystal tunable filter; lauffer et al use a single camera and filter wheel to collect bright field and different band fluorescence images; the research adopts a single-light-path microscopic imaging technology, and the filtering speed is improved by an electric control liquid crystal tunable filter and a filter rotating wheel mode, but the liquid crystal response and the mechanical rotation still have second-level delay, so that the problem of accurate matching of bright field and fluorescent images is difficult to effectively solve.
Disclosure of Invention
Aiming at the problems that phytoplankton cells with similar forms are difficult to distinguish, the impurities with the same particle size are large in interference, the positions of the phytoplankton cells are difficult to locate and divide and the like in the current phytoplankton species identification microscopic examination method, the utility model provides a phytoplankton bright field and fluorescence dual-light path synchronous microscopic imaging device which can directly acquire microscopic bright field images and multiband fluorescence images of phytoplankton in a water body and can be used for rapidly acquiring microscopic multispectral images of the phytoplankton.
The technical scheme of the utility model is as follows: a phytoplankton bright field and fluorescence dual-light path synchronous imaging device, comprising:
the double-light-path imaging optical system is respectively connected to the light source control circuit and the upper computer control system;
the double-light-path imaging optical system comprises a bright-field imaging light path and a fluorescent imaging light path, and the two light paths share a microscopic imaging objective lens and a beam splitting prism;
the light splitting prism is used as a center, the bright field imaging light path adopts transmission type illumination, the fluorescent imaging light path adopts falling type illumination, the light splitting mode after the microscope objective is adopted, and the imaging vision field of the bright field imaging light path is consistent with that of the fluorescent imaging light path;
after the light is split by the microscope objective, the bright field imaging light path and the fluorescent imaging light path are in symmetrical structures by the symmetrical planes of the splitting surfaces of the splitting prism, and the light paths of the two light paths after light splitting reach the respective cameras are consistent.
The bright field imaging light path is sequentially provided with a white light source, a condensing lens, a slide glass, an imaging objective lens collection lens, a beam splitting prism, a bright field cylindrical lens and a bright field camera.
Further, a fluorescent light source, a lens group, a tunable liquid crystal band-pass filter, a transmission beam splitter prism, an imaging objective lens and a slide sample are sequentially arranged on the fluorescent imaging light path, and a dichroic mirror, a narrow-band filter and a fluorescent camera CCD are arranged above the beam splitter prism.
Further, the light source control circuit is used for providing stable driving and controlling the light source switch for different light sources, and comprises: the system comprises a singlechip controller, a constant current source and an analog switch;
the singlechip controller is connected to a constant current source which is connected to a white light source and a fluorescent light source through two paths of analog switches.
Further, the upper computer control system comprises an image acquisition module, an adjusting liquid crystal filter module, a light source selection module and a display module, wherein the image acquisition module is connected to the bright field camera and the fluorescent camera; the liquid crystal filter module is connected to the tunable liquid crystal band-pass filter; the light source selection module is connected to the singlechip controller; the display module is connected with the image acquisition module.
Furthermore, a beam splitting prism is selected and used, and the beam splitting ratio is 3:7.
The utility model has the following advantages:
1. the method comprises the steps of taking a beam splitting prism as a center, designing a phytoplankton bright field and fluorescence dual-light-path synchronous microscopic imaging optical system, wherein transmission type illumination and fluorescence imaging falling type illumination are adopted for bright field imaging; the imaging light path shares a microscopic imaging objective, and a microscopic objective post-light-splitting mode is adopted to ensure the consistency of a bright field and a fluorescent imaging visual field; the split bright field and the fluorescence light path (the position distance of the objective lens, the cylindrical lens and the camera, etc.) are symmetrically structured by the symmetrical plane of the split light surface of the splitting prism, so that the optical paths of the split light paths reaching the respective cameras are consistent, thereby realizing synchronous focusing imaging of the bright field and the fluorescence; the ratio of the reflection light to the transmission light of the light splitting prism is 3:7, namely 30% of the reflected light is used for bright field imaging, and 70% of the transmitted light is used for fluorescence imaging, so that the signal intensity of a fluorescence channel is improved.
Drawings
FIG. 1 shows a phytoplankton bright field and fluorescence dual-light path microscopic imaging optical structure;
FIG. 2 is a dual-path synchronous imaging device structure for phytoplankton.
Reference numerals illustrate: 1. the light source comprises a bright field light source, 2 parts of a condensing lens, 3 parts of a slide plate, 4 parts of an objective lens, 5 parts of a beam splitting prism, 6 parts of a dichroic mirror, 7 parts of a tunable liquid crystal band-pass filter, 8 parts of a lens group, 9 parts of a fluorescent light source, 10 parts of a narrow-band filter, 11 parts of a fluorescent tube lens, 12 parts of a fluorescent camera, 13 parts of a bright field tube lens, 14 parts of a bright field camera, 15 parts of a dual-light-path imaging optical system, 16 parts of an analog switch, 17 parts of a constant current source, 18 parts of a singlechip controller, 19 parts of a light source control circuit, 20 parts of an image acquisition module, 21 parts of a regulating liquid crystal filter module, 22 parts of a light source selection module, 23 parts of a display module and 24 parts of an upper computer control system.
Detailed Description
The technical solutions of the embodiments of the present utility model will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present utility model, and it is apparent that the described embodiments are only some embodiments of the present utility model, but not all embodiments, and all other embodiments obtained by those skilled in the art without the inventive effort based on the embodiments of the present utility model are within the scope of protection of the present utility model.
Aiming at the problems that phytoplankton cells with similar forms are difficult to distinguish, the impurities with the same particle size are large in interference, the positions of the phytoplankton cells are difficult to position and divide and the like in the current phytoplankton species identification microscopic examination method, the utility model adopts a transmission type and epitype combined illumination mode, and designs a phytoplankton bright field and fluorescence dual-light-path synchronous microscopic imaging device by taking a light splitting light path as a core. The device core light path uses the same objective lens to collect light, the bright field imaging light path and the fluorescent imaging light path are separated through the beam splitting prism piece, and the split bright field and fluorescent light path are in symmetrical structures by the symmetrical planes of the beam splitting surfaces of the beam splitting prism, so that the bright field and the fluorescent imaging visual field are ensured to be consistent; the bright field imaging and the fluorescence imaging of the device adopt two independent sets of light sources and cameras.
According to an embodiment of the present utility model, a phytoplankton bright field and fluorescence dual-optical path synchronous imaging device is provided, including:
the double-light-path imaging optical system is respectively connected to the light source control circuit and the upper computer control system;
the double-light-path imaging optical system comprises a bright-field imaging light path and a fluorescent imaging light path, and the two light paths share a microscopic imaging objective lens and a beam splitting prism;
the light splitting prism is used as a center, the bright field imaging light path adopts transmission type illumination, the fluorescent imaging light path adopts falling type illumination, the light splitting mode after the microscope objective is adopted, and the imaging vision field of the bright field imaging light path is consistent with that of the fluorescent imaging light path;
after the light is split after the microscope objective, the bright field imaging light path and the fluorescent imaging light path are symmetrically structured by the symmetrical planes of the splitting surfaces of the splitting prism, so that the light paths of the two light paths after the light is split to the respective cameras are consistent, and the bright field imaging light path and the fluorescent imaging light path are synchronously focused for imaging.
In the embodiment, the utility model uses a beam splitter prism as a center, uses a high-resolution microscopic imaging light path structure, and combines a transmission type illumination mode and an epi-illumination mode to design a phytoplankton bright field and fluorescence dual-light path synchronous microscopic imaging optical system.
Referring to fig. 1, the microscopic imaging optical structure of the phytoplankton bright field and fluorescent light double-light-path comprises a bright field imaging light path and a fluorescent imaging light path, wherein a 10W white light LED is used as a bright field light source 1 in the bright field imaging light path, bright field light source light is focused by a condensing lens 2 and irradiates a slide glass 3, transmitted light is collected by a 40-time imaging objective lens 4, reflected by a beam splitting prism 5 and converged by a bright field cylindrical lens 13, and then received by a bright field camera 14, so that the collection of the bright field image of phytoplankton cells is realized.
The fluorescent imaging light path uses a 50W white light LED as a fluorescent light source 9, after the excitation light lens group 8 is collimated and focused, wavelength selection is carried out through the tunable liquid crystal band-pass filter 7, monochromatic excitation light of 8 wave bands is generated in the range of 440nm to 615nm at intervals of 25nm, the D620nm/660nm dichroic mirror 6 deflects the light direction by 90 degrees, the fluorescent imaging light path passes through the beam splitting prism 5, the imaging objective 4 focuses and irradiates the sample on the glass carrier 3 in an epicenter mode, the sample fluorescence is collected by the imaging objective 4, and the fluorescent imaging light is received by the fluorescent camera 12CCD through the beam splitting prism 5, the dichroic mirror 6 and the BP685/10K narrow-band filter 10, so that the phytoplankton cell imaging of chlorophyll fluorescence wave bands near the 685nm wave bands is realized.
The imaging objective lens uses an objective lens with an infinite correction overlength working distance of 40 times and is matched with a corresponding 200mm focal length cylindrical lens, so that the effective magnification of the system is kept to be 40 times, and clear magnification of phytoplankton cells is realized. In order to make the imaging system reach the optical limit resolution, the resolution of the image obtained by the camera needs to be matched with the resolution of the objective lens, the resolution of the whole imaging is influenced by the maximum resolution of the imaging system and the objective lens, the resolution of the image of the camera of the system needs to be lower than the optical resolution, and the size of the camera phase element is 4.7 mu m.
The light is collected by the same objective lens through the double light paths, then the bright field imaging light path and the fluorescent imaging light path are separated through the beam splitting prism piece, the split bright field and the fluorescent light path (the objective lens, the barrel lens, the camera position distance and the like) are symmetrically structured by the symmetrical plane of the beam splitting surface of the beam splitting prism 5, and the light paths of the two split light paths reach the respective cameras to be consistent, so that under the synchronous condition of ensuring the independence of the positions of different imaging light paths and the imaging, the consistency of the bright field and the fluorescent imaging visual field is ensured.
The light splitting ratio of the beam splitting prism is 3:7, so that the weak signal fluorescent light path is the transmission surface T and accounts for 70% of imaging light, the bright field light path is the reflection surface R and accounts for 30% of imaging light, the fluorescent collection efficiency and the fluorescent signal intensity are improved, and the fluorescent imaging sensitivity is improved.
It can be seen that, for the dual light path of the utility model, the beam splitter prism is used as the center, the bright field imaging adopts transmission illumination, and the fluorescent imaging adopts epiillumination; the imaging light path shares a microscopic imaging objective, and a microscopic objective post-light-splitting mode is adopted to ensure the consistency of a bright field and a fluorescent imaging visual field; the split bright field and the fluorescence light path (the distance between the objective lens, the cylindrical lens and the camera position, etc.) are symmetrically structured by the symmetrical plane of the split light surface of the splitting prism 5, so that the optical paths of the split light paths reaching the respective cameras are consistent, and synchronous focusing imaging of the bright field and the fluorescence is realized.
Further, referring to fig. 2, the present utility model designs a phytoplankton dual-optical-path synchronous imaging device shown in fig. 2, with the optical structure of the dual optical paths as a core. The apparatus comprises: a dual-light path imaging optical system 15, a light source control circuit 19 and an upper computer control system 24.
Under the control of the light source selection module 22, the singlechip controller 18 receives instructions and switches the bright field light source 1 and the fluorescent light source 9 by controlling the gain of the constant current source 17 and the analog switch 16, and the adjustable liquid crystal filter module 21 controls the tunable liquid crystal band-pass filter 7 to control light sources with different wave bands to enter the light path so as to provide excitation light sources for imaging phytoplankton in different modes. After being collected by the objective lens 4 and split by the beam splitter prism 5, the bright field imaging light enters the bright field camera 14 through the bright field cylindrical lens 13 to be collected, the fluorescent imaging light enters the fluorescent camera 12 to be collected after passing through the dichroic mirror 6, the narrow-band filter 10 and the fluorescent cylindrical lens 11, and finally, the image collection module 20 collects and stores microscopic image results of the phytoplankton and displays the microscopic image results on the display module 23.
Wherein the light source control circuit 19 is used for providing stable driving and controlling the light source switch for different light sources. The light source control circuit adopts a constant current source driving mode, so that the stability of the light source is improved, and the singlechip controller 18 is used for outputting different voltage values to adjust the output current of the constant current source 17, so that the light intensity of different light sources can reach the microscopic imaging requirement; the analog switch 16 is used for controlling the switch of the two paths of light sources, so that the switching of different light sources is realized.
The upper computer control system 24 is composed of an image acquisition module 20, an adjustment liquid crystal filter module 21, a light source selection module 22 and a display module 23. The image acquisition module 20 is responsible for controlling different states of the bright field camera and the fluorescent camera, including the switching of the camera, the shooting and the processing of related images; the adjusting liquid crystal filter module is responsible for outputting different instructions to control the tunable liquid crystal band-pass filter to select different wavelength light sources; the light source selection module 22 is responsible for communication and instruction transmission with the singlechip controller 18; the display module is responsible for the display of imaging results and man-machine interaction design.
While the foregoing has been described in relation to illustrative embodiments thereof, so as to facilitate the understanding of the present utility model by those skilled in the art, it should be understood that the present utility model is not limited to the scope of the embodiments, but is to be construed as limited to the spirit and scope of the utility model as defined and defined by the appended claims, as long as various changes are apparent to those skilled in the art, all within the scope of which the utility model is defined by the appended claims.
Claims (6)
1. A phytoplankton bright field and fluorescence dual-light path synchronous imaging device, comprising:
the double-light-path imaging optical system is respectively connected to the light source control circuit and the upper computer control system;
the double-light-path imaging optical system comprises a bright-field imaging light path and a fluorescent imaging light path, and the two light paths share a microscopic imaging objective lens and a beam splitting prism;
the light splitting prism is used as a center, the bright field imaging light path adopts transmission type illumination, the fluorescent imaging light path adopts falling type illumination, the light splitting mode after the microscope objective is adopted, and the imaging vision field of the bright field imaging light path is consistent with that of the fluorescent imaging light path;
after the light is split by the microscope objective, the bright field imaging light path and the fluorescent imaging light path are in symmetrical structures by the symmetrical planes of the splitting surfaces of the splitting prism, and the light paths of the two light paths after light splitting reach the respective cameras are consistent.
2. The phytoplankton bright field and fluorescence dual-light path synchronous imaging device according to claim 1, wherein:
the bright field imaging light path is sequentially provided with a white light source, a condensing lens, a slide glass, an imaging objective lens collection lens, a beam splitting prism, a bright field cylindrical lens and a bright field camera.
3. The phytoplankton bright field and fluorescence dual-light path synchronous imaging device according to claim 1, wherein:
the fluorescent imaging optical path is sequentially provided with a fluorescent light source, a lens group, a tunable liquid crystal band-pass filter, a beam splitter prism, an imaging objective lens and a slide sample, and a dichroic mirror, a narrow-band filter and a fluorescent camera CCD are arranged above the beam splitter prism.
4. The phytoplankton bright field and fluorescence dual-light path synchronous imaging device according to claim 1, wherein:
the light source control circuit includes: the system comprises a singlechip controller, a constant current source and an analog switch;
the singlechip controller is connected to a constant current source which is connected to a white light source and a fluorescent light source through two paths of analog switches.
5. The phytoplankton bright field and fluorescence dual-light path synchronous imaging device according to claim 1, wherein:
the upper computer control system comprises an image acquisition module, an adjusting liquid crystal filter module, a light source selection module and a display module, wherein the image acquisition module is connected to the bright field camera and the fluorescent camera; the liquid crystal filter module is connected to the tunable liquid crystal band-pass filter; the light source selection module is connected to the singlechip controller; the display module is connected with the image acquisition module.
6. The phytoplankton bright field and fluorescence dual-light path synchronous imaging device according to claim 1, wherein:
the beam splitting ratio of the beam splitting prism is 3:7.
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