CN111024934B - Vibration reduction type microscopic observation method - Google Patents
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- CN111024934B CN111024934B CN201911382534.4A CN201911382534A CN111024934B CN 111024934 B CN111024934 B CN 111024934B CN 201911382534 A CN201911382534 A CN 201911382534A CN 111024934 B CN111024934 B CN 111024934B
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Abstract
The invention relates to the technical field of optical microscopic imaging, in particular to a vibration reduction type microscopic observation method, which comprises the following steps: s100, mounting a microscope for performing imaging experiments and/or electrophysiological experiments on living animals and/or a microscope with a membrane clamp on a main platform; s200, fixing the head of the living animal, supporting the limbs of the living animal by a motion table on a sub-platform which isolates the horizontal vibration of a main platform, and adjusting the position of a microscope to aim at the brain of the living animal to observe an imaging experiment and/or an electrophysiological experiment. The invention avoids mutual interference among various experiments, prevents the membrane microelectrode from separating from cells in the experimental process of the microscope with the patch clamp, improves the success rate of imaging experiments and electrophysiological experiments, and can use a plurality of sub-platforms to carry out a plurality of experiments, thereby improving the experimental efficiency.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging, in particular to a vibration reduction type microscopic observation method.
Background
The living experimental animals are indispensable experimental samples for researching cancer, atherosclerosis, nervous system diseases (such as Alzheimer's disease) and infectious diseases, and commonly used living experimental animals comprise mice, rats, marmosets and the like. In the biological experimental research process, since the living microscopic imaging technology can be used for studying and observing specific cells, gene and molecule expression or interaction relation, tracking target cells and imaging drug curative effect from molecular and cellular level, and evaluating the disease development process, the living microscopic imaging or electrophysiological experiment of the living animal applied with experimental means is generally required.
Because the ultrafast laser light source used for multiphoton fluorescence microscopy is very expensive, most biological imaging laboratories share the light source and improve experimental efficiency by installing multiple microscopes on an optical shock absorption platform. Experiments performed on the same optical shock mount platform included: A. anesthesia live body animal microscopic imaging (microscope without patch clamp) experiment, B. anesthesia live body animal electrophysiological experiment (microscope with patch clamp), C. head fixed and sober live body animal microscopic imaging (microscope without patch clamp) experiment and D. head fixed and sober live body animal electrophysiological experiment (microscope with patch clamp), when carrying out A, B, C and D four kinds of experiments on this optical shock-absorbing platform, because the head of the live body animal is fixed, and the rest of the body, such as four limbs can freely move on the locomotor apparatus, the vibrations that the live body animal produced when moving easily let many microscopes on this ordinary optical shock-absorbing platform also produce vibrations, thereby cause: the experiment process A is not influenced by the living animals in the experiments A and B, and the experiment process A is influenced by the movement of the living animals in the experiments C and D; the experiment process B is not influenced by the living animals in the experiments A and B, and the experiment process B is influenced by the movement of the living animals in the experiments C and D; the C experiment process is not influenced by the living animals in the A and B experiments, and the C experiment process is influenced by the movement of the living animals in the C and D experiments; the D experiment process is not influenced by the living animals in the A and B experiments, and the D experiment process is influenced by the movement of the living animals in the C and D experiments.
A microscope with patch clamp used in electrophysiological experiments is a device for recording electrophysiological signals while imaging brain functions in neuroscience research, the brain function imaging is mainly aimed at imaging brain nerve cell activity, because living animals can generate transmembrane flow of calcium ions when brain nerve cells are excited, the calcium ions can change color when meeting calcium ion sensitive dye, thereby being capable of imaging, and the transmembrane flow of calcium ions can cause the potential of brain nerve cells to change, therefore, the electrophysiological signal recording makes the small area cell membrane contacted with the opening of the electrode tip electrically separate from the surrounding cell membrane by contacting the membrane microelectrode with the cell membrane of a cell and sealing with high impedance to achieve the goal of fixing the point position of the cranial nerve cell, the purpose of monitoring and recording the ion current of the ion channel of the cell membrane at the fixed point position is achieved.
The existing living body microscopic imaging device for living animals comprises an optical shock absorption platform for placing a microscope, an object stage, a laser type microscope and a laser light source, wherein when the existing optical shock absorption platform is used, horizontal shock of the optical shock absorption platform caused by movement of the living animals in a plurality of experimental processes can cause mutual interference of a plurality of microscopes on the same optical shock absorption platform, so that the shock of an observation image of a neighboring microscope installed on the same optical shock absorption platform can influence the observation effect, the horizontal shock can cause the shock of the microscope with a patch clamp, so that the patch microelectrode is very easy to separate from a cell membrane, the patch microelectrode needs to be contacted with the cell membrane again, the patch microelectrode can be contacted with a corresponding cell through a plurality of experiments, and the previous same cell is difficult to find again for experiments after the patch microelectrode is separated from the cell membrane, the failure rate of the experiment is increased.
Disclosure of Invention
The invention aims to provide a vibration reduction type microscopic observation method to solve the problem that the experiment fails because a living animal moves in the experiment process.
The vibration damping type microscopic observation method in the scheme comprises the following steps:
s100, mounting a microscope for performing imaging experiments and/or electrophysiological experiments on living animals and/or a microscope with a membrane clamp on a main platform;
s200, fixing the head of the living animal, supporting the limbs of the living animal by a motion table on a sub-platform which isolates the horizontal vibration of the main platform, and adjusting the position of the microscope to align the brain of the living animal to observe an imaging experiment and/or an electrophysiological experiment.
The beneficial effect of this scheme is:
when the cranial nerve cell observation of imaging experiment and/or electrophysiological experiment to living body animal, the main platform for installing microscope is separated from the sub-platform for installing the motion table for moving living body animal, the main platform can not vibrate during the movement of living body animal to cause the vibration of microscope on the main platform, the horizontal vibration which can not be isolated becomes the vertical vibration which can be isolated, the imaging experiment and electrophysiological experiment to living body animal on the main platform can not be influenced, the mutual interference among various experiments is avoided, the separation of cell from the microelectrode of the microscope with patch clamp in the experiment process is prevented, the success rate of imaging experiment and electrophysiological experiment is improved, meanwhile, a plurality of sub-platforms can be used for carrying out a plurality of experiments, and the experiment efficiency is higher.
Further, in step S200, the sliding base slides up and down along the adjusting seat of the main platform, so that the sliding base drives the objective lens soaked with liquid on the horizontal supporting portion to observe the brain of the living animal.
The beneficial effects are that: before the observation of brain nerve cells, the position of the objective lens is adjusted, and liquid capable of increasing amplification factor is soaked on the objective lens, so that the imaging is clearer, and the operation of electrophysiological experiments is facilitated.
Further, in step S200, before the limbs of the living animal are supported on the motion table, the moving plate on the surface plate of the sub-platform is adjusted, so that the motion ball on the limiting plate above the moving plate is located under the microscope, and the limiting plate is driven by the lifting mechanism on the moving plate to adjust the height of the motion ball.
The beneficial effects are that: before a brain nerve cell observation experiment, the moving plate and the lifting mechanism are adjusted to enable the moving ball bearing the living animal to be located at a proper position, so that the living animal is ensured to be located in a range which can be observed by a microscope.
Further, the method comprises a step S300 of adjusting the height of the electrophysiological object stage carrying the micromanipulator and the membrane microelectrode by the adjusting mechanism to make the through hole of the bearing plate on the top of the adjusting mechanism surround the outside of the sport ball.
The beneficial effects are that: the position of an electrophysiological objective table of an electrophysiological experiment is adjusted, the position of a patch microelectrode is limited, and the observation of brain nerve cells in the electrophysiological experiment process is facilitated.
Further, in step S300, the electrophysiology stage is detachably connected to the sub-platform through the adjusting mechanism.
The beneficial effects are that: the electrophysiology objective table does not occupy the position of main platform, can set up a sub-platform to every experiment, can not occupy the too big operating space of main platform, avoids the main platform too in a jumble.
Further, in step S300, the electrophysiology stage is detachably connected to the main platform through the adjusting mechanism.
The beneficial effects are that: the electrophysiology objective table is positioned on the main platform, and the living animal and the sports ball are positioned on the sub-platform, so that horizontal vibration is isolated, and mutual interference is prevented.
Further, the method comprises a step S400 of putting the head of the living animal into a U-shaped frame seat opening of the electrophysiology objective table, screwing a limiting rod on the end part of the frame seat opening, enabling the limiting rod to abut against the head of the living animal from two sides for fixing, and connecting the isolation board to the upper part of the limiting rod and enabling an operation opening on the isolation board to be aligned with the brain of the living animal.
The beneficial effects are that: carry on spacingly in live body animal's brain both sides to carry on spacingly to the top of live body animal brain through the division board, avoid the removal of live body animal brain to cause the experiment failure.
Further, in the step S400, the mouth is put into the hollow tube on the bottom of the recess of the mount while the head of the living animal is positioned in the mount opening.
The beneficial effects are that: the mouth of the living animal is limited by the hollow tube, so that the living animal is prevented from biting a person, meanwhile, oxygen or anesthetic gas can be directly introduced from the hollow tube to carry out an experiment, the state whether the living animal is awake or not can be changed in real time in the experiment process, and the experiment is more convenient.
Further, in step S200, the moving plate is magnetically adsorbed on the surface of the sub-platform, the position of the moving plate is adjusted by the adjusting bolt fixed on the adjusting frame on the sub-platform, and the moving plate is moved on the sub-platform to change the position by the adjusting bolt.
The beneficial effects are that: the movable plate is adsorbed on the sub-platform through magnetism, and the front, back, left and right positions are adjusted through adjusting bolts on the adjusting frame, so that the stability is high.
Further, in the step S300, the electrophysiological object stage is detachably connected through the rectangular array of bolt holes formed on the surface plate body of the sub-platform or the main platform.
The beneficial effects are that: different experimental facilities can be dismantled or installed repeatedly to the bolt hole on sub-platform or the main platform, and it is more convenient to use.
Drawings
FIG. 1 is a block flow diagram of a first embodiment of a vibration damping microscopy method of the present invention;
FIG. 2 is a schematic structural diagram of a vibration damping platform according to a first embodiment of the vibration damping microscopic observation method of the present invention;
FIG. 3 is a top view of the moving plate of FIG. 2;
FIG. 4 is a top view of the securing mechanism of FIG. 2;
fig. 5 is a schematic structural view of a vibration reduction platform in a second embodiment of the vibration reduction type microscopic observation method of the present invention.
Detailed Description
The following is a more detailed description of the present invention by way of specific embodiments.
Reference numerals in the drawings of the specification include: the device comprises a main platform 1, an adjusting seat 2, a horizontal supporting part 3, an objective lens 4, a sliding seat 5, a laser source 6, a sub-platform 7, an electrophysiological objective table 8, a membrane microelectrode 9, a limiting plate 10, a sport ball 11, a moving plate 12, a lifting mechanism 13, an adjusting mechanism 14, a fixing mechanism 15, an adjusting frame 16, an adjusting bolt 17, a frame seat 18, a hollow tube 19, a limiting rod 20, an isolating plate 21 and an operation port 22.
Example one
In order to implement the vibration damping type microscopic observation method of the first embodiment, the first embodiment further discloses a vibration damping platform, as shown in fig. 2: including a main platform 1 and a plurality of sub-platform 7 of keeping apart the 1 horizontal vibrations of main platform, when carrying out the imaging experiment of live body animal, can place the microscope that carries out imaging experiment or electrophysiological experiment or take the microscope of diaphragm pincers to the live body animal on the main platform 1, laser source 6 installs on main platform 1, when carrying out the imaging experiment and the electrophysiological experiment of live body animal simultaneously, place the microscope or take the microscope of diaphragm pincers simultaneously on main platform 1, install the motion platform that supplies the motion of live body animal four limbs on sub-platform 7, the motion platform is placed the live body animal and is supplied the microscope to carry out imaging experiment and/or electrophysiological experiment.
Set up a plurality of bolt holes that become the rectangular array on the panel body of main platform 1 and sub-platform 7, main platform 1 is last to be connected with through the bolt, nut and bolt hole can be dismantled and adjust seat 2, sliding connection has slide 5 on adjusting seat 2, slide 5 is the upper and lower to the sliding height of adjusting seat 2, slide 5 is the U-shaped, threaded connection has the screw thread post on the wall of slide 5 opening both sides, the screw thread post fastens the position of slide 5 on adjusting seat 2, the welding has horizontal support portion 3 on slide 5, horizontal support portion 3 is the cuboid form, the joint has objective 4 on the tip of horizontal support portion 3 keep away from slide 5 one side, soak liquid on the objective 4 terminal surface, liquid can be clear water or oil.
The motion platform includes the motion ball 11, can adjust moving plate 12 and the elevating system 13 of height-adjusting of distance all around, elevating system 13 bottom is located moving plate 12 through screw threaded connection, and the welding of elevating system 13 top has rectangular form limiting plate 10, and motion ball 11 normal running fit is at limiting plate 10 tip, and the normal running fit of motion ball 11 goes on through the pivot, for example sets up the rotation mouth that the diameter is greater than motion ball 11 on the limiting plate 10 tip, and the pivot welding is on the motion mouth, clearance fit between pivot and motion ball 11.
As shown in fig. 3, the moving plate 12 is magnetically adsorbed on the sub-platform 7, and can be adsorbed by embedding a permanent magnet in the moving plate 12, an adjusting frame 16 is sleeved on the periphery of the moving plate 12, the adjusting frame 16 is adhered to the sub-platform 7 for fixing, a plurality of adjusting bolts 17 are connected to the adjusting frame 16 in a threaded manner, and the adjusting bolts 17 are located at the end portions of the adjusting frame 16 to abut against the moving plate 12.
Still including the electrophysiology objective table 8 who bears micromanipulator and diaphragm microelectrode 9, a plurality of screw holes have been seted up on the electrophysiology objective table 8, the rectangular array is arranged into to the screw hole, electrophysiology objective table 8 is located main platform 1, electrophysiology objective table 8 includes a plurality of adjustment height's adjustment mechanism 14, the available piston pneumatic cylinder of adjustment mechanism 14, electrophysiology objective table 8 can dismantle the connection on main platform 1 with the screw mode in adjustment mechanism 14 bottom, the welding of adjustment mechanism 14's top has the loading board, the loading board welds on piston pneumatic cylinder's piston promptly, the through-hole that holds sport ball 11 is seted up at the loading board middle part.
As shown in fig. 4, the fixing mechanism 15 for fixing the head of the living animal is welded on the electrophysiology objective table 8, the fixing mechanism 15 includes a U-shaped frame seat 18, the frame seat 18 is welded on the electrophysiology objective table 8, two end portions of an opening of the frame seat 18 are respectively in threaded connection with a limiting rod 20, a hollow tube 19 for the mouth of the living animal to extend into is welded on the recessed bottom of the opening of the frame seat 18, anesthetic gas or oxygen can be introduced into the hollow tube 19, a partition plate 21 located above the limiting rod 20 is clamped on the frame seat 18, and an operation port 22 capable of aligning with the brain of the living animal is formed in the partition plate 21.
The vibration damping type microscopic observation method, as shown in figure 1, comprises the following steps:
s100, installing a microscope for performing imaging experiments and/or electrophysiological experiments on living animals and/or a microscope with a membrane clamp on a main platform 1;
s200, fixing the head of the living animal, supporting the limbs of the living animal by a motion table on a sub-platform 7 which isolates the horizontal vibration of the main platform 1, and adjusting the position of a microscope to aim at the brain of the living animal to observe an imaging experiment and/or an electrophysiological experiment;
s300, detachably connecting the electrophysiological objective table 8 to the main platform 1 through an adjusting mechanism 14, detachably connecting the electrophysiological objective table 8 through a rectangular array of bolt holes formed in a surface plate body of the main platform 1, adjusting the height of the electrophysiological objective table 8 bearing the micromanipulator and the membrane microelectrode 9 through the adjusting mechanism 14, and enabling a through hole of a bearing plate on the top of the adjusting mechanism 14 to surround the outer side of the sport ball 11;
s400, the head of the living animal is placed into the U-shaped opening of the frame seat 18 of the electrophysiological objective table 8, when the head of the living animal is positioned in the opening of the frame seat 18, the mouth of the living animal is placed into the hollow tube 19 at the bottom of the recess of the frame seat 18, the limiting rod 20 at the end of the opening of the frame seat 18 is screwed, the limiting rod 20 abuts against the head of the living animal from two sides to be fixed, the isolation plate 21 is clamped above the limiting rod 20, and the operation port 22 on the isolation plate 21 is aligned with the head of the living animal.
In the step S200, the slide base 5 is slid up and down along the adjustment seat 2 of the main platform 1, the slide base 5 drives the objective lens 4 soaked with liquid on the horizontal support portion 3 to observe the brain of the living animal, before the limbs of the living animal are supported on the motion table, the moving plate 12 on the surface plate of the sub platform 7 is adjusted, the moving ball 11 on the limiting plate 10 above the moving plate 12 is positioned right below the microscope, the lifting mechanism 13 on the moving plate 12 drives the limiting plate 10 to adjust the height of the moving ball 11, the moving plate 12 is magnetically adsorbed on the surface of the sub platform 7, the position of the moving plate 12 is adjusted by the adjustment bolt 17 fixed on the adjustment frame 16 on the sub platform 7, and the adjustment bolt 17 pushes the moving plate 12 to move on the sub platform 7 to change the position.
In the first embodiment, the experiment of the microscopic imaging (microscope without patch clamp) of the anesthetized living animal, the experiment of the electrophysiological of the anesthetized living animal (microscope with patch clamp), the experiment of the microscopic imaging (microscope without patch clamp) of the fixed-head and conscious living animal, and the experiment of the electrophysiological of the fixed-head and conscious living animal (microscope with patch clamp) are taken as examples, and the cranial nerve cells of the mouse are observed.
Placing a corresponding microscope on a main platform 1, placing four mice required by an experiment on a moving ball 11 of four sub-platforms 7 respectively, adjusting the height of the moving ball 11 and the mice on the moving ball by a lifting mechanism 13, screwing an adjusting bolt 17 on an adjusting frame 16 to adjust the front, back, left and right positions of a moving plate 12, driving the moving ball 11 and the mice on the moving plate 12 to adjust the front, back, left and right positions, fixing the heads of the mice by a frame seat 18 of a fixing mechanism 15, placing mouths of the mice into a hollow tube 19, rotating a limiting rod 20, fixing the heads of the mice from two sides of the heads of the mice by the end part of the limiting rod 20 facing the inner side of the opening of the frame seat 18, clamping a separation plate 21 above the limiting rod 20, aligning an operation port 22 to an operation area of the heads of the mice, and introducing anesthetic gas into the hollow tube 19 of the mice required to be anesthetized.
The sliding slide 5 adjusts the height of the objective 4 in the imaging experiment, so that the objective 4 can clearly image the cranial nerve cells of the head of the mouse, the piston type hydraulic cylinders are synchronously started to drive the micromanipulator and the membrane microelectrode 9 on the bearing plate to adjust the height, and the sport ball 11 and the mouse on the sport ball are kept to be exposed from the through hole.
In the experimental process, the anesthetized mouse can not move, the non-anesthetized conscious mouse can move on the moving ball 11, the moving ball 11 rolls under the action of the mouse, the mouse and the moving ball 11 only can enable the independent sub-platform 7 to vibrate, the sub-platform 7 is independent of the main platform 1, the main platform 1 can not vibrate due to the movement of the mouse and the rolling of the moving ball 11, the imaging experiment and the electrophysiological experiment of the conscious and head-fixed mouse can not interfere with each other, the imaging experiment and the electrophysiological experiment of the anesthetized mouse can not interfere with each other, the horizontal vibration which cannot be isolated is changed into the vertical vibration which can be isolated, the imaging experiment and the electrophysiological experiment of the mouse by the main platform 1 can not be influenced, the mutual interference between the anesthetizing mouse experiment and the conscious and head-fixed mouse experiment can be avoided, the membrane microelectrode 9 of the microscope with the membrane clamp can be prevented from separating from the cell in the experimental process, the success rate of imaging experiments and electrophysiological experiments is improved, and meanwhile, a plurality of sub-platforms 7 can be configured on one main platform 1, so that the experiment efficiency is improved.
Example two
The difference from the first embodiment is that, as shown in fig. 5, the electrophysiological object stage 8 is arranged on the sub-platform 7, the electrophysiological object stage 8 is detachably connected to the sub-platform 7 at the bottom of the adjusting mechanism 14 by means of screws, in the experiment, the head of the mouse is fixed by the frame seat 18 of the fixing mechanism 15, the relative position of the patch microelectrode 9 and the head is not changed due to the fixation of the head of the mouse, therefore, the movement of the mouse and the rotation of the sport ball 11 do not affect the patch microelectrode 9, and sub-platform 7 keeps apart main platform 1 with the motion of mouse and the roll of sport ball 11, can reach the work that prevents mutual interference between a plurality of experiments, and electrophysiology objective table 8 is located sub-platform 7, reduces that micro-operator and diaphragm microelectrode 9 occupy main platform 1 operating space too big among the electrophysiology experiment process, and the main platform 1 of being convenient for carries out a plurality of experiments usefulness.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (8)
1. A vibration damping type microscopic observation method is characterized by comprising the following steps:
s100, when an imaging experiment and an electrophysiological experiment of a living animal are performed simultaneously, mounting a microscope for performing the imaging experiment and the electrophysiological experiment on the living animal or a microscope with a membrane clamp on a main platform;
s200, fixing the head of the living animal, enabling limbs of the living animal to be supported by a motion table on a sub-platform which isolates the horizontal vibration of a main platform, adjusting a movable plate on a surface plate of the sub-platform before supporting the limbs of the living animal on the motion table, enabling a motion ball on a limiting plate above the movable plate to be located under a microscope, driving the limiting plate to adjust the height of the motion ball by a lifting mechanism on the movable plate, and adjusting the position of the microscope to align with the brain of the living animal to carry out imaging experiments and/or observation of electrophysiological experiments;
s300, the height of the electrophysiological objective table bearing the micromanipulator and the membrane microelectrode is adjusted through the adjusting mechanism, so that the through hole of the bearing plate on the top of the adjusting mechanism surrounds the outer side of the sport ball.
2. A vibration damping microscopy method according to claim 1, characterized in that: in step S200, the slide base slides up and down along the adjusting seat of the main platform, so that the slide base drives the objective lens soaked with liquid on the horizontal supporting portion to observe the brain of the living animal.
3. A vibration damping microscopy method according to claim 1, characterized in that: in step S300, the electrophysiology stage is detachably connected to the sub-platform through the adjustment mechanism.
4. A vibration damping microscopy method according to claim 1, characterized in that: in the step S300, the electrophysiology stage is detachably connected to the main platform through the adjusting mechanism.
5. The vibration damping microscopy method according to claim 3 or 4, wherein: the method further comprises a step S400 of putting the head of the living animal into a U-shaped frame seat opening of the electrophysiology objective table, screwing a limiting rod on the end part of the frame seat opening, enabling the limiting rod to abut against the head of the living animal from two sides for fixing, clamping the isolation board above the limiting rod and enabling an operation opening on the isolation board to align to the brain of the living animal.
6. A vibration damping microscopy method according to claim 5, characterized in that: in step S400, the mouth is placed into the hollow tube on the bottom of the recess of the holder when the head of the living animal is positioned in the holder opening.
7. The vibration damping microscopy method according to claim 3 or 4, wherein: in the step S200, the moving plate is magnetically adsorbed on the surface of the sub-platform, the position of the moving plate is adjusted by the adjusting bolt fixed on the adjusting frame on the sub-platform, and the moving plate is moved on the sub-platform to change the position by the adjusting bolt.
8. The vibration damping microscopy method according to claim 3 or 4, wherein: in the step S300, the electrophysiological objective table is detachably connected through the rectangular array of bolt holes formed in the surface plate body of the sub-platform or the main platform.
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