CN116773451A - Imaging auxiliary device, imaging method of biological tissue sample and laser imaging system - Google Patents
Imaging auxiliary device, imaging method of biological tissue sample and laser imaging system Download PDFInfo
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- CN116773451A CN116773451A CN202310767396.1A CN202310767396A CN116773451A CN 116773451 A CN116773451 A CN 116773451A CN 202310767396 A CN202310767396 A CN 202310767396A CN 116773451 A CN116773451 A CN 116773451A
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- 238000003384 imaging method Methods 0.000 title claims abstract description 155
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000010586 diagram Methods 0.000 claims abstract description 18
- 230000003287 optical effect Effects 0.000 claims description 23
- 210000004907 gland Anatomy 0.000 claims description 17
- 230000000007 visual effect Effects 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 4
- 238000003825 pressing Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 229920001821 foam rubber Polymers 0.000 description 3
- 238000009434 installation Methods 0.000 description 3
- 239000012780 transparent material Substances 0.000 description 3
- 239000007769 metal material Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The application relates to an imaging auxiliary device, an imaging method of a biological tissue sample and a laser imaging system, wherein the imaging auxiliary device comprises: a sample carrying device for placing a biological tissue sample to be detected and a positioning and observing device for observing the position of the biological tissue sample; the horizontal plane of the sample bearing device is provided with a scale diagram for indicating the position information of the target object on the horizontal plane; the positioning observation device is used for observing the biological tissue sample in the sample bearing device before imaging and determining the position information of the biological tissue sample in the sample bearing device according to the scale map; the position information is used for controlling an objective lens of a laser imaging system to be rapidly aligned with the biological tissue sample in the imaging process; according to the technical scheme, the rapid imaging of the laser imaging system can be realized, and the imaging efficiency is improved.
Description
Technical Field
The application relates to the technical field of optics, in particular to an imaging auxiliary device, an imaging method of a biological tissue sample and a laser imaging system.
Background
The multiphoton fluorescence technology is widely used for precisely imaging a biological tissue sample on the premise of no damage to the biological tissue sample in the imaging process, and can completely save various original biological information in the biological tissue sample.
Because biological tissue samples are various and different in size, the size of the field of view (FOV) of the multiphoton precision imaging equipment based on the multiphoton fluorescence technology is limited, and is generally far smaller than the size of the biological tissue samples, and changes generated by the direct placement on a sample table along with time are difficult to accurately predict, when the tissue samples are imaged by adopting multiphoton means in situ, imaging blurring occurs frequently, the focal plane time for locking the target z-axis direction is long, and the focal point of the field of view is difficult to quickly determine in the corresponding xy focal plane, particularly, for some biological tissue samples with small sizes (generally within tens to hundreds of cells), such as animal embryos, and the like, because the size of the focal point is far smaller than the bottom area of a sample bearing device, a great amount of time is required to find the position of the biological tissue samples on the sample bearing device during imaging, so that the imaging operation efficiency is greatly influenced.
Disclosure of Invention
Based on this, it is necessary to provide an imaging assisting device, an imaging method of a biological tissue sample and a laser imaging system to improve the imaging operation efficiency in view of at least one of the above technical drawbacks.
An imaging assistance apparatus comprising: a sample carrying device for placing a biological tissue sample to be detected and a positioning and observing device for observing the position of the biological tissue sample;
the horizontal plane of the sample bearing device is provided with a scale diagram for indicating the position information of the target object on the horizontal plane;
the positioning observation device is used for observing the biological tissue sample in the sample bearing device before imaging and determining the position information of the biological tissue sample in the sample bearing device according to the scale map;
wherein the position information is used for controlling an objective lens of a laser imaging system to be rapidly aligned with the biological tissue sample in the imaging process.
In one embodiment, the scale map comprises: a co-ordinate ruler or grid with a spatially visual reference scale.
In one embodiment, the positioning and observing device comprises an optical microscope for searching out the biological tissue sample through an ocular lens and determining the position information of the biological tissue sample in the sample carrying device according to the scale map.
In one embodiment, the positioning and observing device comprises: the camera and the control module connected with the camera; the camera is arranged above a sample table of the laser imaging system;
the camera is used for shooting pictures on the horizontal plane of the sample bearing device;
the control module is used for analyzing the pictures to determine the position information of the biological tissue sample in the sample bearing device.
In one embodiment, the sample carrier device comprises: the sample box, the base, the fixed cover and the scale assembly;
the sample box comprises a culture dish and a gland, the scale chart is arranged on a scale assembly, and the scale assembly is connected to the bottom of the base;
wherein the culture dish is used for bearing biological tissue samples;
the gland structure is matched with the culture dish and is used for covering the culture dish;
the base is used for placing the sample box;
the fixing cover is used for fixing the sample box on the base.
In one embodiment, the scale assembly is a groove design, and the base is mounted to the scale assembly in an embedded manner;
the side of the base is provided with positioning beads, and the side of the scale assembly is provided with positioning holes; wherein, the location pearl matches the location with the locating hole.
In one embodiment, the sample cartridge is of circular structural design;
the bottom of the culture dish and the bottom of the gland are in a plane shape;
a round hollowed-out part is arranged in the middle of the base and used for placing the sample box;
the gland is tightly contacted with the culture dish in an embedded manner;
and a sponge cushion is also arranged at the contact part between the gland and the culture dish.
In one embodiment, the upper portion of the gland is provided with a handle structure for assisting in manipulating the gland to compact the biological tissue sample onto the bottom plane of the culture dish.
In one embodiment, the sample cartridge is a structure made of a transparent material;
the base and the fixed cover are made of metal materials.
In one embodiment, the sample cartridge is a single-use component and the base and stationary cover are reusable components.
In one embodiment, the fixing cover and the base are connected through threads.
A method of imaging a biological tissue sample, comprising:
(1) Placing a biological tissue sample into a sample carrier; wherein, the horizontal plane of the sample bearing device is provided with a staff gauge;
(2) Initial observation is carried out on the sample carrying device, and a target area of the biological tissue sample in the sample carrying device is determined according to the scale diagram;
(3) And placing the sample bearing device on a sample stage of a laser imaging system, controlling an objective lens of the laser imaging system to be aligned with the target area, and rapidly imaging the biological tissue sample.
In one embodiment, the step (2) includes:
placing the sample carrier on a stage of an optical microscope;
searching a biological tissue sample through an ocular lens of an optical microscope;
observing that the biological tissue sample corresponds to a target region of the ruler;
the step (3) comprises:
and leading the target area into control software of a laser imaging system, guiding an objective lens of the laser imaging system to focus on the target area through the control software, and rapidly imaging the biological tissue sample.
In one embodiment, the step (2) includes:
placing the sample carrier on a sample stage of a laser imaging system;
shooting a picture of the chassis range of the sample carrying device by using a camera arranged on the sample table, and analyzing the picture to determine a target area of the biological tissue sample in the sample carrying device;
the step (3) comprises:
and leading the target area into control software of a laser imaging system, guiding an objective lens of the laser imaging system to focus on the target area through the control software, and rapidly imaging the biological tissue sample.
A laser imaging system, comprising: an optical system, a sample stage, a laser imaging device; wherein the sample stage is used for placing the sample carrying device;
in the imaging process, the optical system controls the objective lens to be rapidly aligned with the biological tissue sample in the sample carrying device according to the position information, and irradiates laser on the biological tissue sample; the laser imaging device collects fluorescence generated on the biological tissue sample to image the biological tissue sample.
According to the imaging auxiliary device, the imaging method of the biological tissue sample and the technical scheme of the laser imaging system, the biological tissue sample to be detected is placed by designing the sample bearing device provided with the scale map, and before imaging, the position of the biological tissue sample is observed by utilizing the positioning observation equipment to obtain the position information of the biological tissue sample on the horizontal plane of the sample bearing device; the object lens of the laser imaging system is controlled to be aligned with the biological tissue sample rapidly in the imaging process by utilizing the position information, so that the rapid imaging of the laser imaging system can be realized, and the imaging efficiency is improved.
In addition, the sample bearing device provided by the application is suitable for various multiphoton precise imaging equipment, can be used for bearing various biological tissue samples, and is convenient for quickly locking the positions of the biological tissue samples, so that the imaging operation efficiency is further improved, and the risk of pollution of the biological tissue samples is reduced.
Drawings
FIG. 1 is a schematic illustration of the "light slice" effect of multiphoton fluorescence;
FIG. 2 is an exemplary diagram of an imaging assistance apparatus of one embodiment;
FIG. 3 is a schematic diagram of an exemplary xy coordinate scale;
FIG. 4 is a schematic diagram of an exemplary grid;
FIG. 5 is a schematic diagram of a positioning and viewing device of an embodiment;
FIG. 6 is a schematic diagram of a positioning and viewing device of another embodiment;
FIG. 7 is an exploded view of a sample carrier of one embodiment;
FIG. 8 is a schematic view of a sample carrier in an exemplary separated state;
FIG. 9 is a schematic view of a sample carrier in an exemplary assembled state;
FIG. 10 is a flow chart of a method of imaging a biological tissue sample according to one embodiment;
fig. 11 is a schematic diagram of an exemplary laser imaging system configuration.
Detailed Description
The present application will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
It should be noted that, due to space limitation, all technical solutions cannot be enumerated, only some embodiments are provided in the following examples, and the "first" and "second" described in the present application are used only to distinguish different objects, and have no substantial distinguishing meaning.
In the imaging process of a laser imaging system of the multiphoton fluorescence technology, imaging of a specific focal plane where a focal point is located is generally realized through a galvanometer element, as shown in fig. 1, fig. 1 is a schematic diagram of an effect of optical slicing of multiphoton fluorescence, as shown in the figure, a black point position is a focal point position, an imaging mode of a whole biological tissue sample is realized in a layer-by-layer mode through imaging of each focal plane in an accumulated mode, the imaging is realized through a technical means called optical slicing, in view of the limitation of the size of a field of view (FOV) of a multiphoton precision imaging device, in view of the fact that an objective lens needs to accurately irradiate laser on the biological tissue sample in the imaging process, and because part of the small volume of the biological tissue sample needs to be searched for a long time to be capable of positioning the biological tissue sample, in order to realize positioning of the biological tissue sample, rapid imaging acquisition of the biological tissue sample is facilitated, and imaging operation efficiency is improved.
Referring to fig. 2, fig. 2 is an exemplary diagram of an imaging assistance apparatus of an embodiment, including: a sample carrier 01 and a positioning observation device 02; the sample carrying device 01 is used for placing a biological tissue sample M to be detected, and the sample carrying device 01 is provided with a scale chart on a horizontal plane, so that the position information of a target object on the horizontal plane can be indicated; the positioning observation device 02 observes the position of the biological tissue sample M, observes the biological tissue sample M on the sample bearing device 01 before imaging, and determines the position information of the biological tissue sample M in the sample bearing device 01 according to the scale map; after the position information is obtained, the objective lens 31 of the laser imaging system 03 is controlled to be aligned with the biological tissue sample M in an auxiliary manner in the imaging process, so that rapid imaging can be performed, and the imaging efficiency of the laser imaging system 03 is improved.
In one embodiment, for the scale on the sample carrier 01, it may be a coordinate scale or grid with a spatial visual reference scale, as shown in fig. 3, fig. 3 is a schematic diagram of an exemplary xy coordinate scale, by which the position of the target object in the sample carrier 01 may be indicated, as the hatched area in the figure is the biological tissue sample M, by which the position of the biological tissue sample M in the sample carrier 01 may be determined. As shown in fig. 4, fig. 4 is a schematic diagram of an exemplary grid, scales are marked in different sorting modes in the longitudinal direction and the transverse direction of the grid, and a, b, c … … are marked in the transverse direction, 0, 1, 2 … … are marked in the longitudinal direction, so that the position of the biological tissue sample M in the sample carrier 01 can be determined; for grids, each grid cell can also be distinguished by a different color.
In one embodiment, as shown in fig. 5, fig. 5 is a schematic diagram of a positioning and observing apparatus of one embodiment, where the positioning and observing apparatus 02 may use an optical microscope 21 to search for a biological tissue sample M through an eyepiece 211, and determine position information (such as coordinate parameters) of the biological tissue sample M in the sample carrier 01 according to the scale, and since the optical microscope 21 may observe a fine biological tissue sample M, initial positioning of the position of the biological tissue sample M may be completed; specifically, the focal position (e.g., the exact center of the biological tissue sample M) required for imaging can be visually determined under certain conditions, and the corresponding coordinate parameters (x, y) can be recorded.
After the position information is obtained, the position information is imported into control software of the laser imaging system 03, and a scale image on the sample bearing device 01 is prestored in the control software of the laser imaging system 03, so that a target area can be quickly aligned according to the position information, and further, quick imaging can be assisted; preferably, an optical microscope 21 with a digital output function may be used, where the optical microscope 21 is connected to the laser imaging system 03, and the positional information of the initially positioned biological tissue sample M is imported into the control software of the laser imaging system 03.
In another embodiment, as shown in fig. 6, fig. 6 is a schematic diagram of a positioning and observing device of another embodiment, where the positioning and observing device 02 of this embodiment includes a camera 22 and a control module 23 connected with the camera 22, where the camera 22 is disposed above a sample stage 32 of a laser imaging system 03, and it should be noted that, in a specific implementation, an installation position of the camera 22 may be selected according to different field conditions, and is not limited to a specific installation position; the camera 22 is used for taking pictures on the horizontal plane of the sample carrying device 01; the control module 23 is used for analyzing the pictures to determine the position information of the biological tissue sample M in the sample carrier device 01.
The control module 23 of this embodiment may be connected to the laser imaging system 03, before imaging, the sample carrier 01 is placed on the sample stage 32, the control software of the laser imaging system 03 is utilized to output instructions to the control module 23, the camera 22 is started to take pictures of the sample carrier 01, the position information (such as coordinate parameters) of the biological tissue sample M in the sample carrier 01 can be determined through analysis of the pictures, the scale map on the sample carrier 01 is pre-stored in the control software of the laser imaging system 03, and the target area can be quickly aligned according to the position information, so that rapid imaging can be assisted.
With respect to the sample carrier 01, specific embodiments thereof are described below with reference to the drawings.
Fig. 7 is an exploded view of a sample carrier 01 according to an embodiment, fig. 8 is a schematic view of the sample carrier 01 in an exemplary separated state, and as shown, the sample carrier 01 according to the present embodiment includes: a sample cartridge 10, a scale assembly 11, a base 12, and a fixed cover 13; the sample box 10 comprises a culture dish 101 and a gland 102, the sample box 10 is made of transparent materials, the culture dish 101 is used for bearing biological tissue samples M, and the structure of the gland 102 is matched with the culture dish 101; the scale component 11 is connected with the bottom of the base 12, and the scale component 11 can be made of transparent materials; the scale assembly 11 is marked with a scale pattern at a hollowed-out position relative to the base 12, and the position of the biological tissue sample M on the culture dish 101 is assisted to be positioned through the scale pattern.
When in use, firstly, a biological tissue sample M to be imaged is put into the culture dish 101 to cover the culture dish 101; the base 12 is used for placing the sample box 10, after the biological tissue sample M is placed, the sample box 10 is integrally placed on the base 12, then the sample box 10 is fixed by the fixing cover 13, and finally the base 12 is placed on the scale assembly 11; after preparation is completed, the sample carrier device 01 is placed on the sample stage 32 for imaging; before imaging, scanning a biological tissue sample M on a sample carrying device 01 through a positioning observation device 02, and rapidly positioning the position information of the biological tissue sample M on a culture dish 101 in an auxiliary manner according to a scale chart; during imaging, the corresponding area of the position parameter is used as a light source focus of the laser imaging system 03, the objective lens 31 is controlled to rapidly determine a visual field focus in an xy focal plane and perform focus locking, laser is irradiated on the biological tissue sample M through the objective lens 31, fluorescence generated on the biological tissue sample M is collected, and finally the biological tissue sample M is imaged.
For the sample box 10, it can be circular structural design, and the centre of base 12 is equipped with circular fretwork portion, just can place sample box 10, then fixes sample box 10 through fixed lid 13 for it keeps firm. Further, the bottom of the culture dish 101 and the bottom of the pressing cover 102 are in a plane shape, and the pressing cover 102 is closely contacted with the culture dish 101 in an embedded manner; in order to place the biological tissue sample M on the bottom plane of the culture dish 101, a sponge pad 103 is further placed at the contact position between the pressing cover 102 and the culture dish 101, and the sponge pad 103 may press the biological tissue sample M flat against the bottom plane of the culture dish 101.
In this embodiment, the sample box flattens the biological tissue sample M at the bottom of the culture dish 101 through the gland 102 and the foam-rubber cushion 103, and through the compacting function, the nonstandard biological tissue sample M with an oversized z-axis can be compacted to the z-axis height specified by the sample box 10, so as to shorten the time for locking the focal plane in the z-axis direction of the target, better correct the scanning image cavitation phenomenon caused by the uneven sample, and reduce the frequent occurrence of imaging blurring in the imaging process.
For the scale assembly 11, which may preferably be a groove design, the base 12 is mounted to the scale assembly 11 in an embedded manner; in order to facilitate disassembly, installation and fixation, a positioning bead 121 is arranged on the side edge of the base 12, a positioning hole 111 is arranged on the side surface of the scale assembly 11, and matching positioning is carried out through the positioning bead 121 and the positioning hole 111; when in use, the base 12 is placed into the groove of the scale assembly 11 and fixed, and only the base 12 is required to be pulled out when in disassembly.
In one embodiment, the upper portion of the gland 102 is provided with a handle structure 102a by which the gland 102 may be assisted in compacting the biological tissue sample M onto the bottom plane of the culture dish 101. The sample box 10 can be a disposable part, and is made of disposable materials, so that the biological tissue sample M is prevented from contacting other objects, is well isolated from the laser imaging system 03, the risk of pollution of the biological tissue sample M is reduced, the occurrence of internal and external pollution is avoided, and the requirements of biological safety protection measures are met. The base 12 and the fixed cover 13 are reusable components, and the base 12 and the fixed cover 13 can be made of metal materials; preferably, the fixed cover 13 is in threaded connection with the base 12. In use, after the biological tissue sample M is placed in the sample box 10, the sample box 10 is placed in the hollowed-out portion of the base 12, a protruding portion can be provided in the sample box 10, a clamping groove is provided in the base 12, the protruding portion is clamped into the clamping groove to fix the position, and then the fixing cover 13 is rotated to tightly compress the sample box 10 on the base 12 in a screw-thread pushing manner, so that the biological tissue sample M is prevented from being affected in the moving process.
As in the solution of the above embodiment, the base 12 and the fixed cover 13 can be reused, and the disposable sample cartridge can be matched, so that the risk of pollution can be avoided, and the practicability and the use effect can be ensured.
The sample carrier 01 provided by the application is detachably combined, has a compact structure and is easy and convenient to operate, as shown in fig. 9, fig. 9 is a schematic structural view of the sample carrier 01 in an exemplary combined state, and in a use state, the sample carrier 01 has a very compact structure and can quickly replace the sample cartridge 10; the sample box 10 is disposable, so that good isolation protection of the biological tissue samples M is realized, pollution is prevented, and various biological tissue samples M (such as under a lens, puncture biopsy, embryo and the like) can be accommodated; the base 12, the fixed cover 13 and the scale assembly 11 are provided for repeated use, and the stable base 12 and the fixed cover 13 can stably place the sample cartridge 10 on the sample stage 32, avoiding any slight movement during the imaging process.
The sample carrying device provided by the embodiments can be used for carrying various biological tissue samples, and the position of the biological tissue sample on the culture dish is quickly and assisted to be positioned through the scale pattern marked by the scale component, so that the position of the biological tissue sample is quickly locked by the assisted laser imaging system, and the imaging operation efficiency is improved. Meanwhile, the sample box of the sample bearing device is disposable, so that the contact between the biological tissue sample and other objects is avoided, the risk of pollution of the biological tissue sample is reduced, and the related requirements of biological safety protection measures are met. The biological tissue sample is flattened at the bottom of the culture dish through the gland and the foam-rubber cushion, so that the time for locking the focal plane in the direction of the target z axis is shortened, and the phenomenon of imaging blurring frequently occurs in the imaging process is reduced.
An example of a method of imaging a biological tissue sample is set forth below.
Referring to fig. 10, fig. 10 is a flowchart of a method of imaging a biological tissue sample according to one embodiment, comprising the steps of:
(1) Placing a biological tissue sample M into a sample carrier 01; wherein, the horizontal plane of the sample carrying device 01 is provided with a scale chart.
As shown in fig. 7 to 9, the present application provides an embodiment of a sample carrier device 01, the sample carrier device 01 comprising: the sample box 10, the scale assembly 11, the base 12, the fixed cover 13 and other components; wherein the sample cartridge 10 comprises a culture dish 101 and a gland 102.
Illustratively, step (1) may comprise the following:
s1, firstly placing a biological tissue sample M to be imaged into a culture dish 101;
s2, placing a foam pad 103 on the biological tissue sample M;
s3, placing the gland 102 to cover the culture dish 101, pressing the foam-rubber cushion 103 to press the biological tissue sample M flatly onto the bottom plane of the culture dish 101;
s4, integrally placing the sample cartridge 10 on the base 12, and fixing the sample cartridge by using the fixing cover 13;
s5, the base 12 is placed on the scale assembly 11, and the preparation is completed.
For the scale map, it may be a coordinate ruler or grid with a space vision reference scale, etc., and of course, other forms of scale map may be used to mark the position information.
(2) An initial observation of the sample carrier device 01 is performed, and a target area of the biological tissue sample M in the sample carrier device 01 is determined according to the scale map.
In this step, the positioning observation device 02 may be used to perform initial observation of the sample carrier 01, and to position the target area of the biological tissue sample M in the sample carrier 01 according to the scale of the sample carrier 01.
In one embodiment, step (2) may include the following:
the sample carrier 01 is placed on the stage of the optical microscope 21, and the biological tissue sample M is searched out through the eyepiece 211 of the optical microscope 21, and the biological tissue sample M is observed to correspond to the target area of the scale map.
Specifically, the operator may place the sample carrier 01 on the stage of the optical microscope 21, observe and search the biological tissue sample M through the eyepiece 211, and determine the position information corresponding to the biological tissue sample M through the observation scale map, such as the coordinate scale, so as to obtain the target area where the biological tissue sample M is located.
In another embodiment, step (2) may include the following:
placing the sample carrier device 01 on the sample stage 32 of the laser imaging system 03; a picture of the extent of the chassis of the sample carrier 01 is taken with the camera 22 mounted on the sample stage 32, and the picture is analyzed to determine the target region of the biological tissue sample M in the sample carrier 01.
Specifically, the image includes image contents of the biological tissue sample M and the ruler, an image area of the biological tissue sample M can be obtained by analyzing the image, and then the image area corresponds to the ruler, for example, the position of the mesh where the image area is located can be determined, so that the position information of the biological tissue sample M in the sample carrying device 01 can be determined, and a target area where the biological tissue sample M is located can be obtained.
(3) The sample carrier device 01 is placed on the sample stage 32 of the laser imaging system 03, the objective lens 31 of the laser imaging system 03 is controlled to be aligned with the target area, and the biological tissue sample M is rapidly imaged.
Specifically, according to the target area obtained by initial positioning in the step (2), during the process of imaging the biological tissue sample M by the laser imaging system 03, the objective lens 31 can be controlled to rapidly image the target area.
In one embodiment, when the optical microscope 21 is used, the corresponding procedure of step (3) may include: the target area is guided into control software of the laser imaging system 03, and the objective lens 31 of the laser imaging system 03 is guided to focus on the target area by the control software so as to rapidly image the biological tissue sample M.
In addition, if the camera 22 is used to take a picture, the corresponding procedure of step (3) may include: the target area is guided into control software of the laser imaging system 03, and the objective lens 31 of the laser imaging system 03 is guided to focus on the target area by the control software so as to rapidly image the biological tissue sample M.
Embodiments of a laser imaging system are set forth below.
As shown in fig. 11, fig. 11 is a schematic structural diagram of an exemplary laser imaging system, including: comprising the following steps: an optical system 30, a sample stage 32, a laser imaging device 33; wherein the sample stage 32 is used to place the sample carrier device 01 in any embodiment of the application.
Prior to imaging, the sample carrier 01 may be initially observed with the positioning observation device 02 and positioned to a target region of the biological tissue sample M in the sample carrier 01 according to a scale of the sample carrier 01. For example, the operator can determine the positional information corresponding to the biological tissue sample M by the optical microscope 21, thereby obtaining the target area where the biological tissue sample M is located.
Further, a camera 22 may be further disposed on the sample stage 32 of the laser imaging system 03, where the camera 22 is connected to control software of the laser imaging system 03 through a control module 23, and the control software may output instructions to control the camera 22 to take a picture of a chassis range of the sample carrier 01, where the control module 23 may analyze the picture by using a related image processing algorithm, or after the control module 23 performs a certain process on the picture, send image data to the control software to process the image data, and the control software may implant a related algorithm to analyze the image data, so as to determine a target area of the biological tissue sample M in the sample carrier 01.
In the imaging process, the position information of the target area is imported into control software of the laser imaging system 03, and the optical system 30 controls the objective lens 31 to be rapidly aligned with the biological tissue sample M in the sample carrying device 01 according to the position information, so that the laser irradiates the biological tissue sample M; the laser imaging device 33 collects fluorescence generated on the biological tissue sample M to image the biological tissue sample M.
According to the technical scheme, the sample bearing device suitable for the multiphoton precise imaging equipment is designed, can be used for bearing various biological tissue samples, and then is placed on the sample table for imaging detection, so that the position of the biological tissue samples can be conveniently and rapidly locked, and the imaging operation efficiency is improved. Meanwhile, the risk of pollution of the biological tissue sample is reduced, the time for locking the focal plane in the z-axis direction of the target is shortened, the focal point of the visual field is rapidly determined in the xy focal plane, and the phenomenon of imaging blurring frequently occurs in the imaging process is reduced.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (10)
1. An image forming assist device, comprising: a sample carrying device for placing a biological tissue sample to be detected and a positioning and observing device for observing the position of the biological tissue sample;
the horizontal plane of the sample bearing device is provided with a scale diagram for indicating the position information of the target object on the horizontal plane;
the positioning observation device is used for observing the biological tissue sample in the sample bearing device before imaging and determining the position information of the biological tissue sample in the sample bearing device according to the scale map;
wherein the position information is used for controlling an objective lens of a laser imaging system to be rapidly aligned with the biological tissue sample in the imaging process.
2. The imaging assistance apparatus according to claim 1, wherein the scale map includes: a co-ordinate ruler or grid with a spatially visual reference scale.
3. The imaging assistance apparatus according to claim 1, wherein the positioning observation device includes an optical microscope for searching out a biological tissue sample through an eyepiece and determining positional information of the biological tissue sample in a sample carrier from the scale map.
4. The imaging assisting apparatus according to claim 1, wherein the positioning observation device includes: the camera and the control module connected with the camera; the camera is arranged above a sample table of the laser imaging system;
the camera is used for shooting pictures on the horizontal plane of the sample bearing device;
the control module is used for analyzing the pictures to determine the position information of the biological tissue sample in the sample bearing device.
5. The imaging support apparatus of any of claims 1-4, wherein the sample carrier apparatus comprises: the sample box, the base, the fixed cover and the scale assembly;
the sample box comprises a culture dish and a gland, the scale chart is arranged on a scale assembly, and the scale assembly is connected to the bottom of the base;
wherein the culture dish is used for bearing biological tissue samples;
the gland structure is matched with the culture dish and is used for covering the culture dish;
the base is used for placing the sample box;
the fixing cover is used for fixing the sample box on the base.
6. An imaging assistance apparatus as claimed in claim 5 wherein the scale assembly is of a recessed design, the base being mounted to the scale assembly in an embedded manner;
the side of the base is provided with positioning beads, and the side of the scale assembly is provided with positioning holes; wherein, the location pearl matches the location with the locating hole.
7. A method of imaging a biological tissue sample, comprising:
(1) Placing a biological tissue sample into a sample carrier; wherein, the horizontal plane of the sample bearing device is provided with a staff gauge;
(2) Initial observation is carried out on the sample carrying device, and a target area of the biological tissue sample in the sample carrying device is determined according to the scale diagram;
(3) And placing the sample bearing device on a sample stage of a laser imaging system, controlling an objective lens of the laser imaging system to be aligned with the target area, and rapidly imaging the biological tissue sample.
8. The imaging assisting apparatus according to claim 7, wherein the step (2) includes:
placing the sample carrier on a stage of an optical microscope;
searching a biological tissue sample through an ocular lens of an optical microscope;
observing that the biological tissue sample corresponds to a target region of the ruler;
the step (3) comprises:
and leading the target area into control software of a laser imaging system, guiding an objective lens of the laser imaging system to focus on the target area through the control software, and rapidly imaging the biological tissue sample.
9. The imaging assisting apparatus according to claim 7, wherein the step (2) includes:
placing the sample carrier on a sample stage of a laser imaging system;
shooting a picture of the chassis range of the sample carrying device by using a camera arranged on the sample table, and analyzing the picture to determine a target area of the biological tissue sample in the sample carrying device;
the step (3) comprises:
and leading the target area into control software of a laser imaging system, guiding an objective lens of the laser imaging system to focus on the target area through the control software, and rapidly imaging the biological tissue sample.
10. A laser imaging system, comprising: an optical system, a sample stage, a laser imaging device; wherein the sample stage is for placing the sample carrier of any one of claims 1-6;
in the imaging process, the optical system controls the objective lens to be rapidly aligned with the biological tissue sample in the sample carrying device according to the position information, and irradiates laser on the biological tissue sample; the laser imaging device collects fluorescence generated on the biological tissue sample to image the biological tissue sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310767396.1A CN116773451A (en) | 2023-06-27 | 2023-06-27 | Imaging auxiliary device, imaging method of biological tissue sample and laser imaging system |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310767396.1A CN116773451A (en) | 2023-06-27 | 2023-06-27 | Imaging auxiliary device, imaging method of biological tissue sample and laser imaging system |
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| Publication Number | Publication Date |
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| CN116773451A true CN116773451A (en) | 2023-09-19 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202310767396.1A Pending CN116773451A (en) | 2023-06-27 | 2023-06-27 | Imaging auxiliary device, imaging method of biological tissue sample and laser imaging system |
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| Country | Link |
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| CN (1) | CN116773451A (en) |
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- 2023-06-27 CN CN202310767396.1A patent/CN116773451A/en active Pending
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