CN212255074U - Multi-channel fluorescence full-field scanning imaging device - Google Patents
Multi-channel fluorescence full-field scanning imaging device Download PDFInfo
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- CN212255074U CN212255074U CN202020647021.3U CN202020647021U CN212255074U CN 212255074 U CN212255074 U CN 212255074U CN 202020647021 U CN202020647021 U CN 202020647021U CN 212255074 U CN212255074 U CN 212255074U
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Abstract
The utility model provides a multichannel fluorescence full field of vision scanning image device, include: the device comprises an optical path microscopic imaging device, a sample bracket and a displacement objective table; the displacement objective table comprises an X-axis moving mechanism and a Y-axis moving mechanism, the X-axis moving mechanism and the Y-axis moving mechanism respectively drive the displacement objective table to move along the X-axis direction and the Y-axis direction, so that a sample in a sample bracket is driven to move, the part of the sample to be detected exposed under the microscopic field of view of the light path microscopic imaging device is changed, the part of the sample to be detected exposed under the microscope objective lens is adjusted, and the full-field-of-view scanning of the sample to be detected is realized. The utility model discloses avoid tissue slice or unicellular suspension incomplete field of vision scanning and shoot under the microscopic magnification state and lead to the unable concatenation in later stage complete and the image information loss that causes.
Description
Technical Field
The utility model relates to a belong to life science field, specifically, relate to a multichannel fluorescence full field of vision scanning imaging device, especially a multichannel fluorescence full field of vision scanning imaging device of tissue slice or unicellular suspension.
Background
Planar scanning imaging plays an important role in the study of both pathological tissue sections and single cell suspensions. Tissue slice imaging is a powerful tool for observing the examination results of histological structures and analyzing spatial information of cells. However, the imaging field of a microscope, particularly a high power microscope, is limited, and a slice can only be photographed and imaged at a local position under the magnification of the microscope, and cannot be observed for a complete tissue slice in one field, so that the overall appearance of a sample cannot be embodied, and some instruments can photograph a plurality of fields in order to increase the sampling amount, but are not continuous, so that the complete splicing cannot be performed, or due to the reason of mechanical precision, images are overlapped or gapped among the images, so that the imaging of the tissue slice is incomplete, and thus spatial information of important cells in the sample may be lost. For single cell suspensions, the fluorescent full-field scanning imaging is used for accurately counting cells, and comprises the number of cells in a fluorescent image under the dyeing of a live cell dye and a fluorescent image under the dyeing of a dead cell dye, so as to obtain the total cell number and the ratio of the live cells. The current cell counting instrument also has the defects that the sample can not be completely exposed under a microscope objective lens in one visual field, and the cell distribution is uneven and local cell agglomeration is easily caused due to cell division or cell adhesion in a cell suspension, so that the accuracy of the cell counting instrument is seriously influenced.
Through the discovery of retrieval, chinese patent with application number CN201811198222.3 discloses an artifical count and calibration equipment based on image recognition method cell count appearance, including full-automatic cell count appearance, full-automatic cell count appearance includes the main control system, the main control system has a man-machine interface, this main control system has complete machine hardware control module auto focus module and automatic image recognition module, still include a camera, show little light path group, fluorescence filter block module, objective lens module, fluorescence light source and lens group, automatic sample platform and visible light source and lens group, this full-automatic cell count appearance still is provided with an electronic objective table and a change draw-in groove mechanism, the inside mainboard of host computer inputs a virtual groove software package and manual calculation software, and use the cell count board of multiple type. In the above patent, the electric stage is moved to ensure that the counting area of the apparatus is the same as that of the blood cell counting plate, so that the full-field cell counting cannot be realized, and the accuracy of the cell counting needs to be further improved.
SUMMERY OF THE UTILITY MODEL
To the defect among the prior art, the utility model aims at providing a multichannel fluorescence full field of vision scanning imaging device realizes carrying out full field of vision scanning to the sample that awaits measuring, has effectively improved the integrality of the sample formation of image that awaits measuring and the accuracy of follow-up cell count.
In order to achieve the above object, the present invention provides a multi-channel fluorescence full-field scanning imaging device, comprising: the light path microscopic imaging device is used for the multipath fluorescence microscopic imaging of a sample to be detected and is used for fixing a sample bracket at the position of the sample to be detected; the displacement object stage is used for bearing the sample bracket; wherein the content of the first and second substances,
the displacement stage includes:
the X-axis moving mechanism drives the displacement object stage to move along the X-axis direction;
the Y-axis moving mechanism drives the displacement object stage to move along the Y-axis direction;
the X axis and the Y axis are positioned on the same horizontal plane, and the horizontal plane is vertical to the light path of the light path microscopic imaging device; the displacement object stage moves in the X-axis and Y-axis directions to drive the sample to be detected in the sample bracket to move, so that the part of the sample to be detected exposed under the microscopic view of the light path microscopic imaging device is changed, the part of the sample to be detected exposed under the microscope objective lens is adjusted, and the full-view scanning of the sample to be detected is realized.
Preferably, the multi-path fluorescence full-field scanning imaging device comprises a picture acquisition mechanism, wherein the picture acquisition mechanism is arranged at an imaging end of the light path micro-imaging device and is used for acquiring a picture of the sample to be detected exposed in the micro-field of the light path micro-imaging device after the displacement object stage moves each time, so that multiple pictures of the whole sample to be detected exposed in the micro-field of view are obtained.
Preferably, the X-axis moving mechanism includes a first power component and a first transmission component, the first power component is connected to the first transmission component, the first power component drives the first transmission component to move in the X-axis direction, and the first transmission component drives the displacement stage to move in the X-axis direction.
Preferably, the Y-axis moving mechanism includes a second power component and a second transmission component, the second power component is connected to the second transmission component, the second power component drives the second transmission component to move in the Y-axis direction, and the second transmission component drives the displacement stage to move in the Y-axis direction.
Preferably, the multi-path fluorescence full-field scanning imaging device comprises an electric control mechanism, the electric control mechanism comprises a first control component and a second control component, the input ends of the first control component and the second control component are connected with the output end of the control mechanism, and the output ends of the first control component and the second control component are respectively connected with the input ends of the first power component and the second power component, wherein the first control component controls the first power component to drive the first transmission component, and the first transmission component drives the displacement stage and the sample bracket to move, so as to control the movement displacement of the sample bracket in the X-axis direction; the second control component controls the second power component to drive the second transmission component, and the second transmission component drives the displacement object stage and the sample bracket to move, so that the movement displacement of the sample bracket in the Y-axis direction is controlled, and the area of the sample to be detected exposed in the microscopic field of view of the light path microscopic imaging device is adjusted.
Preferably, the first transmission component and the second transmission component have the same structure and both comprise an encoder and a grating ruler, the encoder detects and adjusts the step loss of the first power component or the second power component in time, and the grating ruler is used for measuring the displacement distance and converting a measurement output signal into digital pulses so that the linear distance translated by the displacement object stage in the directions of the X axis and the Y axis is converted into absolute displacement from relative displacement.
Preferably, the first transmission component and the second transmission component comprise a screw rod, a coupler, a bottom plate, a bracket, a guide rail and a slide block, wherein the screw rod is fixed on the bottom plate through the bracket, one end of the screw rod is connected with one end of the coupler, and the other end of the coupler is connected with the first power component or the second power component; the guide rails are arranged on two sides of the screw rod, and two ends of the guide rails are respectively fixed with the bracket; the sliding block is arranged above the screw rod and is movably connected with the guide rails on the two sides, and the screw rod rotates to drive the sliding block to slide along the direction of the guide rails; the grating ruler reading head is arranged in the sliding block and used for measuring the displacement distance recorded by the grating ruler below the sliding block.
Preferably, the multi-channel fluorescence full-field scanning imaging device further comprises: and the Z-axis lifting mechanism is connected with an objective lens of the light path micro-imaging device and drives the objective lens to displace in the vertical direction, so that the distance between the objective lens and the sample to be detected is adjusted, and automatic focusing is realized.
Preferably, the light path microscopic imaging device comprises a bright field light source and a fluorescence field light source, and can respectively obtain microscopic imaging of the sample to be detected under a bright field and a fluorescence field.
Preferably, the fluorescence field light source includes a multi-channel fluorescence light source and a light path switching component, the light path switching component switches the light path of the fluorescence field according to the fluorescence dyeing band feature, the light path switching component is disposed below the multi-channel fluorescence light source, and the light path switching component drives the multi-channel fluorescence light source to move along the horizontal direction, so as to realize the light path switching of the fluorescence field.
Compared with the prior art, the utility model discloses at least one kind's beneficial effect as follows has:
1. the utility model provides an above-mentioned device, through X axle moving mechanism, Y axle moving mechanism drives displacement objective table and sample support groove at the X axle, Y axle direction removes, thereby the adjustment sample that awaits measuring exposes the position under microscope objective, can cover the whole region (whole position) of the sample that awaits measuring through the removal of displacement objective table, obtain the microscopic imaging at a plurality of positions that whole sample that awaits measuring exposes under the microscopic field of vision, realize the full field of vision scanning to the sample that awaits measuring, can avoid the sample that awaits measuring (tissue slice or unicellular suspension) incomplete field of vision scanning and shoot under the microscopic magnification state and lead to the unable concatenation in later stage complete and the image information loss that causes.
2. The utility model discloses the encoder that further sets up and the shift position of grating chi linkage accurate control X axle moving mechanism, Y axle moving mechanism make the displacement objective table change absolute displacement from relative displacement at the linear distance of X axle, Y axle direction translation to accurate control sample that awaits measuring exposes the position under microscope objective, guarantees the perfect whole regions that cover the sample that awaits measuring.
3. The device of the utility model can adjust the distance between the objective lens and the sample to be measured for automatic focusing by controlling the displacement distance of the Z-axis lifting mechanism through the Z-axis lifting mechanism connected with the objective lens; the focusing range is within 0-8mm, and the precision is accurate to 10 mu m, so that high-resolution full-field scanning is realized.
4. The utility model provides an above-mentioned device through multichannel fluorescence light source and light path switching part, can realize many fluorescence detection, can freely switch over a plurality of excitation light sources in the same experimentation, is applicable to the sample that awaits measuring of different fluorescence staining, can accurately judge the cell condition of dying, cell concentration, activity.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic structural diagram of a multi-channel fluorescence full-field scanning imaging device according to a preferred embodiment of the present invention;
FIG. 2 is a schematic structural view of a power unit and a transmission unit according to a preferred embodiment of the present invention;
fig. 3 is an optical path diagram of an optical path micro-imaging device according to a preferred embodiment of the present invention;
the scores in the figure are indicated as: the device comprises a displacement objective table 1, a sample bracket 2, a bright field light source 3, a multi-channel fluorescent light source 4, an optical path microimaging device 5, a picture acquisition mechanism 6, an electric control mechanism 7, a Z-axis lifting mechanism 8, a direct current power supply 9, an encoder 10, a first power part 11, a support 12, a coupler 13, a lead screw 14, a guide rail 15, a sliding block 16, a grating ruler 17 and an optical path switching part 18.
Detailed Description
The present invention will be described in detail with reference to the following embodiments. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that various changes and modifications can be made by one skilled in the art without departing from the spirit of the invention. These all belong to the protection scope of the present invention. Portions not described in detail in the following embodiments may be implemented using existing components.
Referring to fig. 1, it is a schematic structural diagram of a multi-path fluorescence full-field scanning imaging device according to a preferred embodiment of the present invention, and the diagram includes a light path micro-imaging device 5, a sample bracket 2 and a displacement stage 1, where the light path micro-imaging device 5 is used for multi-path fluorescence micro-imaging of a sample to be measured; the sample bracket 2 is used for fixing the position of a sample to be detected, and the sample to be detected can be a tissue slice or a single cell suspension; the displacement stage 1 is used for carrying a sample bracket 2. Also included is a dc power supply 9 for powering the instrument.
The displacement objective table 1 comprises an X-axis moving mechanism and a Y-axis moving mechanism, and the X-axis moving mechanism drives the displacement objective table 1 to move along the X-axis direction. The Y-axis moving mechanism drives the displacement object stage 1 to move along the Y-axis direction; the X axis and the Y axis are positioned on the same horizontal plane, and the horizontal plane is vertical to the light path of the light path micro-imaging device 5. The displacement objective table 1 moves in the directions of the X axis and the Y axis to drive the sample to be detected in the sample bracket 2 to move, so that the part of the sample to be detected exposed under the microscopic view of the light path microscopic imaging device 5 is changed, the part of the sample to be detected exposed under the objective lens of the microscope is adjusted, the whole sample to be detected can be covered by the movement of the displacement objective table 1, the microscopic imaging of a plurality of parts of the whole sample to be detected exposed under the microscopic view is obtained, and the full-view scanning of the sample to be detected is realized.
In other partially preferred embodiments, the multi-channel fluorescence full-field scanning imaging device further comprises a picture acquisition mechanism 6. The image acquisition mechanism 6 is arranged at the imaging end of the light path micro-imaging device 5, and is used for acquiring images of the sample to be detected exposed to the micro-vision field of the light path micro-imaging device 5 after the displacement objective table 1 moves each time, so that a plurality of images of local positions of the whole sample to be detected exposed to the micro-vision field are obtained. Preferably, the image capturing mechanism 6 may be a CCD industrial camera with 2000 ten thousand high definition pixels, or a CMOS camera.
In other preferred embodiments, the X-axis moving mechanism includes a first power component 11 and a first transmission component, the first power component 11 is connected to the first transmission component, the first power component 11 drives the first transmission component to move in the X-axis direction, and the first transmission component drives the displacement stage 1 to move in the X-axis direction.
In some other preferred embodiments, the Y-axis moving mechanism includes a second power unit and a second transmission unit, the second power unit is connected to the second transmission unit, the second power unit drives the second transmission unit to move in the Y-axis direction, and the second transmission unit drives the displacement stage 1 to move in the Y-axis direction.
In other preferred embodiments, the multi-channel fluorescence full-field scanning imaging device further comprises an electric control mechanism 7, the electric control mechanism 7 comprises a first control component and a second control component, and output ends of the first control component and the second control component are respectively connected with input ends of a first power component 11 and a second power component; the first control component controls the first power component 11 to drive the first transmission component, and drives the displacement object stage 1 and the sample bracket 2 to move through the first transmission component, so as to control the movement displacement of the sample bracket 2 in the X-axis direction; the second control part controls the second power part to drive the second transmission part, the second transmission part drives the displacement objective table and the sample support groove 2 to move, and the movement displacement of the sample support groove 2 in the Y-axis direction is controlled, so that the area of the sample to be measured exposed in the field of view of the objective lens is adjusted.
As a preferred embodiment, the electric control mechanism 7 further includes a third control part and a fourth control part, the third control part is used for controlling a third power part of the Z-axis lifting mechanism 8; the fourth control means is for controlling a fourth power means of the optical path switching means.
In other partially preferred embodiments, referring to fig. 2, the first transmission component further comprises a screw 14, a coupling 13, a base plate, three brackets 12, a guide rail 15 and a slider. The rear end of the first power component 11 is connected with the encoder 10, and the front end is connected with the screw rod 14 through the coupling 13 and fixed on the bottom plate through the bracket 12. The bottom plate is a horizontal plate. The three brackets 12 are respectively vertically fixed on the bottom plate to play a supporting role. Guide rails 15 are symmetrically arranged on two sides of the screw rod 14, and the screw rod 14 and two ends of the guide rails 15 are sleeved on the bracket 12 connected with the bottom plate for fixing. The screw rod 14 is screwed with a sliding block 16 movably connected with the guide rails 15 at two sides, the upper part of the sliding block 16 is connected with the lower part of the displacement objective table 1, and a grating ruler 17 is further arranged inside the sliding block 16 for accurately measuring the displacement distance. The rotating speed of the screw rod 14 is effectively changed by controlling the first power component 11, the screw rod 14 rotates to drive the sliding block 16 to slide along the direction of the guide rail 15, the displacement objective table 1 is driven to move, and the sample to be detected can stably and accurately move by controlling the moving distance of the sliding block 16. Through the cooperation of the encoder 10 and the grating ruler 17 in the first power component 11 and the first transmission component, the displacement distance accuracy of the sliding block 16 reaches the level of 10 micrometers, the distance movement of absolute displacement can be realized, the distance error accumulation of multiple movement measurement is avoided, and accurate full-field scanning is achieved. The second transmission component adopts the same structure as the first transmission component, and the structural view of the second transmission component is omitted, and the second transmission component can be specifically referred to the structural view shown in fig. 2.
In other preferred embodiments, referring to fig. 2, the first transmission component and the second transmission component have the same structure and both include an encoder 10 and a grating ruler 17, the encoder 10 detects and adjusts the step loss of the first power component 11 or the second power component in time, and controls the precise rotation of the first power component 11 or the second power component, so as to control the rotation of the screw rod 14 in the first transmission component or the second transmission component and the displacement distance of the slider 16. The reading head of the grating ruler 17 is arranged in the slider 16 and is used for measuring the displacement distance recorded by the grating ruler 17 under the slider. The second transmission member has the same structure as the first transmission member in the present embodiment. The device is used for timely detecting and adjusting the step loss of the second power component and controlling the accurate rotation of the second power component, thereby controlling the rotation of the screw rod 14 in the second transmission component and the displacement distance of the slide block. The grating rulers 17 in the first transmission component and the second transmission component are used for measuring displacement distances of slide blocks on an X axis and a Y axis in a micrometer level, measuring output signals are converted into digital pulses, the digital pulse signals are converted into displacement amounts through the high-speed counting module, the linear distance of the displacement object stage 1 translating in the X axis direction and the Y axis direction is converted into absolute displacement from relative displacement, and full-view scanning of a sample to be measured is realized through the displacement of the displacement object stage 1. The first power component 11 and the second power component can be powered by a motor, such as a stepping motor.
In other parts of the preferred embodiments, referring to fig. 3, the optical path microscopic imaging device 5 comprises a bright field light source 3, an objective lens, a multi-channel fluorescent light source 4, a right-angle prism and a lens barrel; the bright field light source 3 is supported right above the sample bracket 2 through a rod, the displacement object stage 1 is lifted through a support rod, and the bright field light source 3 is used for providing a bright field LED light source. The objective lens is disposed directly below the displacement stage 1. As a preference, a 40-fold objective lens may be employed. The multi-channel fluorescent light source 4 is arranged right below the objective lens and used for providing light source excitation of a fluorescent field. The multi-channel fluorescent light source 4 comprises a fluorescent light source, a fluorescent condenser, an excitation filter, a dichroic mirror and an emission filter, wherein the multi-channel fluorescent light source 4 comprises two or more than two exciters (such as a first exciter and a second exciter) of the fluorescent light source. The fluorescence light source sequentially penetrates through the fluorescence condenser and the excitation filter, and the excitation light is projected onto a sample to be detected under the turning action of the dichroic mirror to form an excitation light path; after the dye in the sample to be detected absorbs the exciting light, the emitted emission light sequentially passes through the dichroic mirror and the emission optical filter through the objective lens to form an emission light path. The right-angle reflecting prism is arranged below the multi-channel fluorescent light source 4 and used for changing the direction of a light path. The working principle of the light path microscopic imaging device 5 is as follows: when the bright field light source is switched on, the fluorescent light source is in a closed state, and the bright field light source passes through the bright field condenser and is projected onto a sample to be detected to form a bright field light path; or the bright field light source is turned off when the fluorescent light source needs to be turned on. The fluorescence light source projects exciting light to a sample to be measured under the turning action of the dichroic mirror through the fluorescence condenser and the optical filter. After the dye in the sample to be detected absorbs the exciting light, the emitted light passes through the dichroic mirror and the emission optical filter through the objective lens, then passes through the 90-degree turning light path of the right-angle reflecting prism, and forms an image through the imaging lens to form a fluorescence field light path.
In other preferred embodiments, the optical path switching component is disposed right below the multi-channel fluorescent light source 4, and performs optical path switching of the fluorescent field according to the fluorescent dyeing waveband feature; the optical path switching member may be fixed to the bottom plate of the entire apparatus by a support rod. In a specific embodiment, the optical path switching member may include a fourth transmission member, a fourth power member; wherein, the input end of the fourth power component can be connected with the output end of the fourth control component of the electric control mechanism 7, and the output end of the fourth power component is connected with the input end of the fourth transmission component. As a preferable mode, the fourth transmission component can adopt the structure of the first transmission component to realize the function thereof, a sliding block of the fourth transmission component is connected with the bottom of the multi-channel fluorescent light source 4, and the sliding block drives the multi-channel fluorescent light source 4 to move in the horizontal direction, so that the light source channels of the multi-channel fluorescent light source 4 are switched, and the light source switching is realized. Through the light path switching component, a plurality of excitation light sources can be freely switched in the same experiment process, and the device is suitable for samples to be detected with different fluorescent stains, for example, the cell apoptosis condition can be accurately judged by adopting Hoechst, AnnexinV, PI and JC-1 stains; cell concentration and activity can be judged by trypan blue staining, calcein-AM/draq-7 and AO/PI double-fluorescence staining.
In specific implementation, the bright field light source 3, the multi-channel fluorescent light source 4, the CCD camera 6, the electric control mechanism 7, and the Z-axis 8 in the above embodiments are electrically connected to a dc power supply 9, respectively.
In other preferred embodiments, the multi-channel fluorescence full-field scanning imaging device further comprises a Z-axis lifting mechanism 8 connected with the objective lens of the light path micro-imaging device 5, and the Z-axis lifting mechanism drives the objective lens to displace in the vertical direction, so as to adjust the distance between the objective lens and the sample to be measured, and adjust the focal length. In a specific embodiment, the Z-axis lifting mechanism 8 may include a third power component and a third transmission component, an input end of the third power component may be connected with an output end of the third control component of the electric control mechanism 7 in the above embodiment, and an output end of the third power component is connected with an output end of the third transmission component. Preferably, the third transmission member may have the same structure as the first transmission member, and a slider of the third transmission member is connected to the objective lens, and the slider drives the objective lens to move linearly in the Z-axis direction. The third control part controls the third power part to drive the third transmission part to move along the Z-axis direction, so that the objective lens is driven to displace in the Z-axis direction, the distance between the objective lens and the sample to be measured is controlled, and the function of adjusting the focal length is achieved. By arranging the Z-axis lifting mechanism 8 connected with the objective lens, the distance between the objective lens and a sample to be measured can be adjusted for automatic focusing by controlling the displacement distance of the Z-axis lifting mechanism 8, the focusing range can be within 0-8mm, and the precision can be accurate to 10 micrometers.
When the above embodiment is implemented specifically, the multi-channel fluorescence full-view scanning imaging device may further be connected to a control mechanism, an auto-focusing control module, a positioning photographing module, an image stitching module, and a counting module.
Wherein the control means is adapted to control the path of movement of the displacement stage 1. The input ends of the X-axis moving mechanism and the Y-axis moving mechanism are connected with the output end of the control mechanism, the control mechanism controls the X-axis moving mechanism and the Y-axis moving mechanism to control the displacement objective table 1 to move according to a set path, the moving distance of the displacement objective table 1 each time is the same as the microscopic magnification visual field of the light path microscopic imaging device 5, and the moving path of the sample to be detected exposed under the microscopic visual field covers the whole sample to be detected.
The autofocus control module can achieve autofocus of the picture taking mechanism 6. The Z-axis lifting mechanism 8 is connected with the input end of the automatic focusing control module, the output end of the automatic focusing control module is connected with the camera (the picture acquisition mechanism 6), the camera is controlled to continuously shoot in the displacement process of the Z-axis lifting mechanism 8 through the movement of the Z-axis lifting mechanism 8, multiple continuously shot images in the displacement process are subjected to resolution analysis, the clearest image is found out, the position of the corresponding image in the Z-axis shooting direction is recorded, and the automatic focusing process is realized.
The positioning photographing module can enable the displacement object stage to move in the X-axis direction and the Y-axis direction to drive a sample to be measured in the sample bracket to move at a fixed distance in an oriented mode and photograph. The output end of the control mechanism is connected with the input end of the positioning photographing module, the output end of the positioning photographing module is connected with the input end of the picture acquiring mechanism 6, the positions of the sample to be measured on the X axis and the Y axis are controlled and dynamically recorded, and when the distance of one microscopic magnification visual field (namely the distance of one objective lens) is moved, the picture acquiring mechanism 6 acquires the picture of the sample to be measured exposed in the microscopic visual field of the light path microscopic imaging device 5, so that multiple pictures of the local positions of the whole sample to be measured exposed in the microscopic visual field are acquired. In the specific implementation: firstly, under a bright field light source, controlling the movement of a sample to be detected, and moving the distance of one objective lens each time to ensure that a plurality of local position pictures of the whole sample to be detected exposed under a microscopic field of view are obtained under the bright field light source; then the fluorescence light source is switched, and the operation is repeated, so that a plurality of local photos of the whole sample to be detected under the condition that the fluorescence light source is exposed in the microscopic field are obtained.
The image splicing module aligns and splices a plurality of images of all the obtained images according to the shooting sequence and integrates the images into a complete full-view image. The output end of the picture acquisition mechanism 6 is connected with the input end of the image Stitching module, and as an optimal mode, the image Stitching module can be stitched into a complete image by adopting a Stitching algorithm in a multi-view image mosaic. The complete image of the sample to be detected can be obtained through the image splicing module, and the problem of cell counting deviation caused by the influence of overlapping or gaps among the images on cell space information is avoided. In specific implementation, after the whole tissue slice or the single cell suspension sample is completely shot by a plurality of pictures under the microscopic field of view of a single light source, the light source is switched while the shot pictures are spliced by the image splicing module, and the sample to be detected is moved at a fixed distance in a fixed direction and shot under another light source, so that the scanning efficiency is accelerated on the premise of ensuring the picture splicing quality.
And the counting module obtains full-field pictures of the single cell suspension under a bright field light source and different fluorescent fields according to the image splicing module, respectively calculates the number of cells in the images of the single cell suspension under the bright field light source, the fluorescent images under the living cell dye dyeing and the fluorescent images under the dead cell dye dyeing, and compares and analyzes the three images to obtain the total cell number, the cell agglomeration rate, the double cell rate and the living cell rate of the single cell suspension. Cell clumping and double cell rates were calculated based on the volume of cells after colony being significantly greater than the volume of a single cell.
In one embodiment, a multi-channel fluorescence full-field scanning imaging device for full-field scanning imaging of tissue slices comprises the following steps:
1. a paraffin-embedded tissue block section was subjected to HE (hematoxylin-eosin) staining, which stained nuclei blue with hematoxylin, and cytoplasm, muscle, connective tissue, and erythrocytes red with eosin.
2. The stained tissue section is placed on the sample bracket 2.
3. The automatic focusing control module adjusts the displacement distance of the Z-axis lifting mechanism 8 to automatically adjust the focal length, and selects the position with the highest definition to focus; then, under a bright field light source, the mixed liquid in the sample bracket 2 is driven to move at a fixed distance in an X-axis direction and a Y-axis direction through the movement of the displacement object stage 1, and a picture is taken, wherein the moving distance of each time is the same as the microscopic magnification visual field, the picture of the mixed liquid exposed to the microscopic visual field of the light path microscopic imaging device 5 after each time of movement of the displacement object stage 1 is obtained, a plurality of local position pictures of the whole mixed liquid exposed to the microscopic visual field are obtained, after the picture is taken, the plurality of shot pictures are aligned and spliced into a complete image through the image splicing module, and the complete image under the bright field light source is obtained; and simultaneously switching to a fluorescent light source, repeating the steps, carrying out directional fixed-distance movement, photographing and picture splicing on the sample to be detected, and finally obtaining a complete image under the fluorescent light source.
The embodiment avoids the influence on the acquisition of cell space information and the accuracy of complete imaging of the tissue section due to overlapping or gaps between the images.
In another embodiment, a multi-channel fluorescence full-field scanning imaging device for full-field scanning imaging of single cell suspension comprises the following steps:
1. after 10 mul of single cell suspension is stained by AO/PI fluorescent reagent, live cells emit green fluorescence by AO staining, and dead cells emit red fluorescence by PI staining.
2. And adding the mixed solution dyed by the AO/PI fluorescent reagent in the step into a sample plate to be detected, and placing the sample plate on a sample bracket 2.
3. The automatic focusing control module adjusts the displacement distance of the Z-axis lifting mechanism 8 to automatically adjust the focal length, and selects the position with the highest definition to focus; then, under a bright field light source, the mixed liquid in the sample bracket 2 is driven to move at a fixed distance in an X-axis direction and a Y-axis direction through the movement of the displacement object stage 1, and a picture is taken, wherein the moving distance of each time is the same as the microscopic magnification visual field, the picture of the mixed liquid exposed to the microscopic visual field of the light path microscopic imaging device 5 after each time of movement of the displacement object stage 1 is obtained, a plurality of local position pictures of the whole mixed liquid exposed to the microscopic visual field are obtained, after the picture is taken, the plurality of shot pictures are aligned and spliced into a complete image through the image splicing module, and the complete image under the bright field light source is obtained; and simultaneously switching to a fluorescent light source, repeating the steps, carrying out directional fixed-distance movement, photographing and picture splicing on the sample to be detected, and finally obtaining a complete image under the fluorescent light source.
After the complete image is obtained in the above embodiment, a counting module can be further connected, and the counting module counts cells and analyzes the cell concentration and the cell activity according to the staining condition of the cells. And generating a result report, namely referring to the cell counting result and information such as cell concentration and cell activity.
In the embodiment, the displacement object stage 1 moves in the directions of the X axis and the Y axis to drive the sample to be measured in the sample bracket 2 to move, so that the full-view scanning of the sample to be measured is realized. The cell counting device can relieve cell counting deviation caused by local cell agglomeration or uneven mixing on a cell counting plate, avoid accumulation of measurement errors and ensure that displacement errors are accurate to within 10 mu m.
The foregoing description of the specific embodiments of the invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by those skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. A multi-channel fluorescence full-field scanning imaging device, comprising: the light path microscopic imaging device is used for the multipath fluorescence microscopic imaging of a sample to be detected and is used for fixing a sample bracket at the position of the sample to be detected; the device is characterized by also comprising a displacement object stage for bearing the sample bracket; wherein the content of the first and second substances,
the displacement stage includes:
the X-axis moving mechanism drives the displacement object stage to move along the X-axis direction;
the Y-axis moving mechanism drives the displacement object stage to move along the Y-axis direction;
the X axis and the Y axis are positioned on the same horizontal plane, and the horizontal plane is vertical to the light path of the light path microscopic imaging device; the displacement object stage moves in the X-axis and Y-axis directions to drive the sample to be detected in the sample bracket to move, so that the part of the sample to be detected exposed under the microscopic view of the light path microscopic imaging device is changed, the part of the sample to be detected exposed under the microscope objective lens is adjusted, and the full-view scanning of the sample to be detected is realized.
2. The multi-channel fluorescence full-field scanning imaging device according to claim 1, further comprising a picture acquiring mechanism, wherein the picture acquiring mechanism is disposed at an imaging end of the optical path micro-imaging device, and is configured to acquire a picture of the sample to be detected exposed to the micro-field of view of the optical path micro-imaging device after each movement of the displacement stage, so as to obtain multiple pictures of the entire sample to be detected exposed to multiple local positions of the micro-field of view.
3. The multi-channel fluorescence full-field scanning imaging device according to claim 1, wherein the X-axis moving mechanism comprises a first power unit and a first transmission unit, the first power unit is connected to the first transmission unit, the first power unit drives the first transmission unit to move in the X-axis direction, and the first transmission unit drives the displacement stage to move in the X-axis direction.
4. The multi-channel fluorescence full-field scanning imaging device according to claim 3, wherein the Y-axis moving mechanism comprises a second power unit and a second transmission unit, the second power unit is connected with the second transmission unit, the second power unit drives the second transmission unit to move in the Y-axis direction, and the second transmission unit drives the displacement stage to move in the Y-axis direction.
5. The multi-channel fluorescence full-field scanning imaging device according to claim 4, further comprising an electric control mechanism, wherein the electric control mechanism comprises a first control unit and a second control unit, the input ends of the first control unit and the second control unit are connected with the output end of the control mechanism, and the output ends of the first control unit and the second control unit are respectively connected with the input ends of the first power unit and the second power unit, wherein the first control unit controls the first power unit to drive the first transmission unit, and the first transmission unit drives the displacement stage and the sample bracket to move, so as to control the movement displacement of the sample bracket in the X-axis direction; the second control component controls the second power component to drive the second transmission component, and the second transmission component drives the displacement object stage and the sample bracket to move, so that the movement displacement of the sample bracket in the Y-axis direction is controlled, and the area of the sample to be detected exposed in the microscopic field of view of the light path microscopic imaging device is adjusted.
6. The multi-channel fluorescence full-field scanning imaging device according to claim 4, wherein the first transmission member and the second transmission member have the same structure and each include an encoder and a grating ruler, the encoder detects and adjusts the step loss of the first power member or the second power member in time, and the grating ruler is used for measuring a displacement distance and converting a measurement output signal into a digital pulse, so that a linear distance of the displacement stage in the X-axis and Y-axis directions is converted from a relative displacement to an absolute displacement.
7. The multi-channel fluorescence full-field scanning imaging device according to claim 6, wherein the first transmission component and the second transmission component further comprise a screw rod, a coupler, a bottom plate, a bracket, a guide rail and a slider, wherein the screw rod is fixed on the bottom plate through the bracket, one end of the screw rod is connected with one end of the coupler, and the other end of the coupler is connected with the first power component or the second power component; the guide rails are arranged on two sides of the screw rod, and two ends of the guide rails are respectively fixed with the bracket; the sliding block is arranged above the screw rod and is movably connected with the guide rails on the two sides, and the screw rod rotates to drive the sliding block to slide along the direction of the guide rails; the grating ruler reading head is arranged in the sliding block and used for measuring the displacement distance recorded by the grating ruler below the sliding block.
8. The multi-channel fluorescence full-field scanning imaging device according to claim 1, further comprising: and the Z-axis lifting mechanism is connected with an objective lens of the light path micro-imaging device and drives the objective lens to displace in the vertical direction, so that the distance between the objective lens and the sample to be detected is adjusted, and automatic focusing is realized.
9. The multi-channel fluorescence full-field scanning imaging device according to any one of claims 1 to 8, wherein the light path microscopic imaging device comprises a bright field light source and a fluorescence field light source, and can respectively obtain microscopic imaging of the sample to be detected under a bright field and a fluorescence field.
10. The multi-channel fluorescence full-field scanning imaging device according to claim 9, wherein the fluorescence field light source comprises a multi-channel fluorescence light source and a light path switching component, the light path switching component switches the light path of the fluorescence field according to the fluorescence dyeing band feature, the light path switching component is disposed below the multi-channel fluorescence light source, and the light path switching component drives the multi-channel fluorescence light source to move along the horizontal direction, so as to realize the light path switching of the fluorescence field.
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| CN112764211A (en) * | 2021-02-04 | 2021-05-07 | 上海睿钰生物科技有限公司 | Microscopic imaging device and microscopic imaging method |
| CN113884489A (en) * | 2021-09-29 | 2022-01-04 | 电子科技大学 | Grating ruler assisted positioning thick liquid layer cell automatic microscopic imaging method |
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| CN112764211A (en) * | 2021-02-04 | 2021-05-07 | 上海睿钰生物科技有限公司 | Microscopic imaging device and microscopic imaging method |
| CN113884489A (en) * | 2021-09-29 | 2022-01-04 | 电子科技大学 | Grating ruler assisted positioning thick liquid layer cell automatic microscopic imaging method |
| CN113884489B (en) * | 2021-09-29 | 2023-06-20 | 电子科技大学 | Automatic microscopic imaging method for thick liquid layer cells with auxiliary positioning of grating ruler |
| CN115078363A (en) * | 2022-08-18 | 2022-09-20 | 广东奥素液芯微纳科技有限公司 | Microfluidic liquid-transfering observation instrument |
| CN115078363B (en) * | 2022-08-18 | 2022-12-02 | 广东奥素液芯微纳科技有限公司 | Micro-fluidic liquid-transfering observation instrument |
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