CN214373782U - Freezing device and detection system for low-temperature degradation imaging of biological sample - Google Patents
Freezing device and detection system for low-temperature degradation imaging of biological sample Download PDFInfo
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- CN214373782U CN214373782U CN202120127069.6U CN202120127069U CN214373782U CN 214373782 U CN214373782 U CN 214373782U CN 202120127069 U CN202120127069 U CN 202120127069U CN 214373782 U CN214373782 U CN 214373782U
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Abstract
A freezing device and a detection system for denudation imaging of a biological sample at low temperature belong to the field of mass spectrum detection and imaging and analysis. The freezing device for the biological sample degradation imaging at the low temperature is characterized in that a top plate clamping groove is formed in a top plate and matched with a bottom plate clamping groove formed in a bottom plate, and a front plate is arranged at the front end of the freezing device to form a cooling and heat dissipation cavity; the semiconductor refrigeration sheet and the water cooling plate in the refrigeration unit are arranged oppositely and tightly connected; one side of the water cooling plate is arranged on the bottom plate; a carrying support plate through hole is further formed in the top plate, the carrying support plate is arranged above the semiconductor refrigerating sheet through the carrying support plate through hole, heat conduction mud is coated on the carrying support plate, and a groove is formed in the middle of the heat conduction mud and used for placing a glass slide of a biological sample; the semiconductor refrigerating sheet is connected with a power supply through a wire; the water cooling plate is communicated with the circulating condensed water tank through a pipeline. The device has the advantages of low price, high cooling speed and low temperature fluctuation, ensures the detection accuracy of the biological sample and has strong freezing capacity.
Description
Technical Field
The utility model belongs to the technical field of mass spectrometric detection and formation of image and analysis, in particular to a refrigerating plant and detecting system that are used for biological sample to degrade formation of image under low temperature.
Background
In recent years, laser ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) has been rapidly developed. Compared with the traditional inductively coupled plasma method, the laser ablation inductively coupled plasma mass spectrometry method reduces a plurality of sample pretreatment steps, and eliminates the polyatomic interference problem caused by water and acid. The surface of the sample is subjected to high-energy ablation through laser to generate aerosol, and the aerosol is carried into the inductively coupled plasma through carrier gas to be detected, so that quantitative analysis is realized. Compared with other technologies, the laser ablation inductively coupled plasma has the advantages of real-time performance, in-situ performance, high resolution, low detection limit, high spatial resolution and the like.
The biological tissue micro-area imaging detection refers to the element content test of a specific micro-area of a biological tissue, and the distribution of the element content in a sample is represented by an image. However, the room temperature ablation has the following disadvantages:
(1) the laser heat effect of the biological sample is often generated in the ablation at the normal temperature, so that the edge of an ablation pit is melted, the fractionation effect is increased, and the precision and the sensitivity of the detection are influenced.
(2) In the dehydration process of the biological sample, metal elements are often lost, so that the defects of laser ablation detection and imaging are caused. Therefore, on the basis of normal-temperature ablation, the frozen sample stage is ablated by using laser for imaging detection of the micro-area of the biological tissue, so that the ablation at low temperature is ensured, and the defect of the ablation at normal temperature is overcome.
The laser ablation frozen sample stage used for the biological tissue micro-area imaging detection has the advantages of reducing the fractionation effect caused by the ablation heat effect, realizing the original form detection of a biological sample, reducing the pollution to the biological sample in the traditional sample preparation process and the like, and becomes an indispensable sample stage for the laser ablation detection in recent years.
Although LA-ICP-MS can achieve better ablation effect under the freezing condition, the existing commercial laser ablation frozen sample table has the advantages of good stability and mature technology, and the technology is also paid more and more attention by scientific researchers. However, the commercial laser ablation frozen sample stage also has the following problems:
(1) the price is high and is over ten thousand.
(2) Commercial laser ablation frozen sample platform, its suitability is poor, is unfavorable for carrying out further technological upgrading repacking.
(3) Which is not conducive to replacing damaged parts.
For the problems, the laser ablation frozen sample table self-made in a laboratory has a large operation range, is beneficial to further improvement and assembly, and is favored to a certain extent. However, the self-made laser ablation frozen sample table reported in the laboratory at present has the following problems:
(1) the operation is complicated by using liquid nitrogen or the like as the refrigerant gas.
(2) The temperature reduction process is slow, which is not beneficial to improving the experimental efficiency.
(3) The stability of the freezing temperature is poor, and the temperature fluctuation is large, so that the precision of the experiment is influenced.
SUMMERY OF THE UTILITY MODEL
To the present existing commercial laser ablation frozen sample platform and the laboratory self-control laser ablation frozen sample platform existing problem, the utility model provides a refrigerating plant and detecting system for biological sample degrades formation of image under the low temperature, this refrigerating plant for biological sample degrades formation of image under the low temperature includes the laser ablation frozen sample platform, circulation condensate tank and the power of self-assembly. The water cooling plate and the semiconductor refrigerating plate are used as core parts in the laser ablation frozen sample stage, and the freezing effect is realized. And the self-assembled laser ablation frozen sample stage is combined in an assembling mode, so that parts damaged inside are easily replaced. And scribbled one deck heat conduction mud on the year thing tray of the frozen sample platform of self-assembling laser ablation, can make biological sample cold more even like this, because heat conduction mud is stronger to the reflex action of light, consequently can make the focus in biological sample field of vision reach the effect unanimous with commercial normal atmospheric temperature sample platform to realize biological sample's accurate focus, avoided setting up the laser light trap in the detection. The utility model discloses a refrigerating plant maximum advantage that is used for biological sample to degrade formation of image under low temperature is the low price, with the utility model discloses an example, the frozen sample platform main part expense of laser degradation of self-assembly is 1700 yuan, and the total price is 4700 yuan, greatly reduced the cost. And the temperature reduction speed is high, the temperature reaches-20 ℃ within 4min, the temperature fluctuation is low, the temperature fluctuation is +/-0.1 ℃ within ten hours, the detection accuracy of the biological sample is ensured, and the freezing capacity is strong.
The utility model relates to a freezing device for biological sample degradation imaging at low temperature, which comprises a laser degradation frozen sample stage, a circulating condensed water tank and a power supply;
the laser ablation frozen sample stage comprises a front plate, a top plate, a bottom plate, a refrigeration unit, a carrying supporting plate and heat conducting mud;
a top plate clamping groove is formed in the top plate, the top plate clamping groove is matched with a bottom plate clamping groove formed in the bottom plate, and a front plate is arranged at the front end of the top plate to form a cooling and heat dissipation cavity;
the refrigerating unit comprises a semiconductor refrigerating sheet and a water-cooling plate; the semiconductor refrigerating plate and the water cooling plate are arranged oppositely and tightly connected; one side of a water cooling plate in the refrigeration unit is arranged on the bottom plate;
a carrying support plate through hole is further formed in the top plate, the carrying support plate is arranged above the semiconductor refrigerating sheet through the carrying support plate through hole, heat conduction mud is coated on the carrying support plate, and a groove is formed in the middle of the heat conduction mud and used for placing a glass slide of a biological sample;
the semiconductor refrigerating sheet is connected with a power supply through a wire; the water cooling plate is communicated with the circulating condensed water tank through a pipeline.
Further, the refrigeration unit is preferably a plurality of units connected in series.
Furthermore, the pipeline connecting the water cooling plate and the circulating condensed water tank is preferably a silicone tube.
Furthermore, the semiconductor refrigeration piece is closely connected with the water cooling plate by adopting heat conduction silica gel to ensure normal contact.
Furthermore, the semiconductor refrigeration piece and the carrying supporting plate are connected through heat conducting silica gel, and the semiconductor refrigeration piece and the carrying supporting plate are used for uniform conduction of refrigeration effects.
The semiconductor refrigerating piece is preferably a C series semiconductor refrigerating piece, and more preferably an XH-C1206 semiconductor refrigerating piece.
Further, the size of the bottom plate is determined according to the size of the refrigeration unit and the size of the degradation pool.
Furthermore, a clamping groove for fixing the refrigeration unit and a pipeline placing guide groove are also arranged on the bottom plate.
Further, the top plate and the bottom plate cooperate to form a pipeline inlet and a pipeline outlet.
Preferably, for convenience of processing and installation, the front plate comprises a first front plate, a second front plate and a third front plate, and the front plate is preferably an aluminum plate and used for fixing the laser ablation frozen sample table.
Preferably, the carrier plates are heat transfer material carrier plates, preferably copper plates. The thermal conductivity of the heat transfer material is more than or equal to 401K (W/m.K).
Preferably, the top plate is a heat-insulating and cold-insulating material top plate, preferably a polytetrafluoroethylene top plate, and is used for heat insulation and cold insulation.
Preferably, the bottom plate is a heat-preservation and cold-insulation material bottom plate, preferably a polytetrafluoroethylene bottom plate, and is used for heat preservation and cold insulation.
Preferably, the circulating condensed water tank is model number LX-300.
Preferably, the power supply is an X271 refrigeration semiconductor dedicated power supply.
A detection system for the denudation imaging of a biological sample at low temperature comprises a refrigerating device, a temperature measuring device, a laser denudation system and an inductively coupled plasma mass spectrometer, wherein the refrigerating device is used for the denudation imaging of the biological sample at low temperature;
the temperature measuring device is used for measuring the temperature of the refrigerating device for the denudation imaging of the biological sample at low temperature;
the laser ablation system is used for ablating the biological sample on the glass slide;
and the inductively coupled plasma mass spectrometer is used for performing mass spectrometry and data processing on the denuded sample.
A method of using a freezing device for imaging degradation of a biological sample at cryogenic temperatures, comprising the steps of:
step 1: after assembling the laser ablation frozen sample table, sealing by using sealant; starting a circulating condensed water tank, adjusting the temperature of the circulating condensed water tank to 5-10 ℃, and stabilizing;
in the step 1, the temperature of the circulating condensed water is stabilized to be more than 10min and the temperature fluctuation is within +/-0.05 ℃.
Step 2: turning on a power supply, starting the semiconductor refrigerating sheet to work, reducing the temperature of the refrigerating unit to minus 50-minus 20 ℃, measuring the temperature of the carrying supporting plate, and waiting for the temperature to be stable;
in the step 2, the temperature stabilizing time is 15-20 min.
And step 3: placing a glass slide loaded with biological sample slices on a carrying pallet, placing a laser ablation frozen sample stage in an ablation solution, and sealing;
in the step 3, in order to increase the sealing performance, an electronic sealant is applied to the slit.
And 4, step 4: introducing helium into the denudation solution, and flushing and removing impurity gas to obtain denudation solution after impurity removal;
and 5: adjusting an online detection mode, wherein the air flow is adjusted to be 50mL/min, and the total air flow is 700 mL;
step 6: setting laser ablation parameters, particularly corresponding laser energy intensity, according to a detected biological sample, and setting plasma mass spectrum detection parameters;
and 7: and starting ablation imaging to obtain a micro-area image of the biological tissue.
The utility model discloses a freezing device and detecting system that is used for biological sample to degrade formation of image under low temperature compares with the frozen sample platform of current laser degradation, the utility model has the advantages of:
1. low in cost, the utility model discloses the overall cost is low, greatly reduced the cost. And the operation is convenient, and the parts damaged by falling, aging and the like are easy to replace.
2. The safety is good, and due to the use of the semiconductor refrigerating sheet, the release and use of the refrigerating gas are reduced, the use cost is reduced, and the subsequent expense of the refrigerating gas is not needed.
3. The freezing time is short, the temperature of the utility model can be reduced to-20 ℃ within 4min, the waiting time of the detection personnel is reduced, and the stable freezing temperature is obtained within about 15 min; the utility model can keep very stable temperature, and the average temperature of 1h-10h is (-26.0 +/-0.1) DEG C.
4. The light holes are not needed, the surface of the carrying tray is coated with heat conduction mud, and a good focusing effect can be obtained through reflection of the heat conduction mud to light.
Drawings
FIG. 1 is a schematic view of an assembly structure of a laser ablation frozen sample stage in a freezing apparatus for ablation imaging of a biological sample at low temperature;
FIG. 2 is an exploded view of a laser ablation frozen sample stage of the freezing apparatus of FIG. 1 for low temperature ablation imaging of a biological sample;
FIG. 3 is a schematic diagram of a freezing apparatus for imaging degradation of a biological sample at low temperatures;
FIG. 4 is a cooling curve of a laser ablation frozen sample stage; (a) a cooling curve graph of a laser ablation frozen sample table is obtained; (b) the temperature profile of the frozen sample stage was laser ablated over 10 hours. The dotted line is an isotherm at-20 ℃;
FIG. 5 is a comparison between a commercial laser ablation normal temperature sample stage and the field focusing effect of the present invention; (a) a focusing effect graph using light-transmitting holes for comparative example; (b) the focusing effect diagram of the heat conduction mud used in the embodiment 1 of the utility model is shown;
in the above figures, 1 is a temperature measuring device; 2 is a power supply; 3 is a circulating condensed water tank; 4 is a silicone tube which is connected with the circulating condensation water tank and the water cooling plate; 5 is a wire which is connected with the semiconductor refrigerating sheet and a power supply; 6 is a first front plate; 7 is a second front plate; 8 is a third front plate; 9 is a bottom plate; 10 is a water cooling plate; 11 is a top plate; 12 is a glass slide; 13 is heat conducting mud; 14 is a semiconductor refrigerating sheet; 15 is a carrying supporting plate; 16 is a pipeline outlet; and 17 is a pipeline inlet.
Detailed Description
The following detailed description of the embodiments of the present invention is made with reference to the accompanying drawings and examples.
In the following embodiments, the freezing device for imaging the biological sample at low temperature mainly matches with the NWR Image 266nm laser ablation of kaelai instruments (element Scientific), and is suitable for TwoVol2 ablation cells, and other types of ablation cells can modify the frozen sample stage according to the size, but the internal refrigeration principle is consistent, and the principle and effect of the present invention are mainly described herein. The utility model discloses can be used to the biological sample's under the freezing condition degradation formation of image.
In the following embodiments, the semiconductor refrigeration piece is an XH-C1206 semiconductor refrigeration piece;
the type of the water cooling plate: pure aluminum wide water-cooled panels;
the heat conduction mud is heat conduction silica gel mud with the heat conductivity coefficient of 6W/m.K.
Example 1
A freezing device for biological sample ablation imaging at low temperature has a structure schematic diagram shown in figure 3, and comprises a laser ablation frozen sample stage, a circulating condensed water tank 3 and a power supply 2;
the structure schematic diagram of the laser ablation frozen sample stage is shown in fig. 1, and the explosion schematic diagram of the laser ablation frozen sample stage is shown in fig. 2, and the laser ablation frozen sample stage comprises three front plates with different sizes, a top plate 11, a bottom plate 9, a refrigeration unit, a carrying pallet 15 and heat-conducting mud 13;
the refrigerating unit comprises an XH-C1206 semiconductor refrigerating piece 14 and a water cooling plate 10 arranged below the semiconductor refrigerating piece 14, wherein the water cooling plate 10 is made of pure aluminum, the semiconductor refrigerating piece 14 is tightly connected with the water cooling plate 10 through heat-conducting silica gel, and the heat-conducting silica gel is also smeared above the semiconductor refrigerating piece 14. The circulating condensed water tank 3 is connected with the water cooling plate 10 by using the silicone tube 4, so that the temperature of the whole water cooling plate 10 is consistent with that of the condensed water. The semiconductor refrigerating sheet 14 is connected with the power supply 2 by the electric wire 5, and after the power is on, the temperature difference between the upper end of the semiconductor refrigerating sheet 14 and the upper end of the water cooling plate 10 can reach 60 ℃, so that the refrigerating effect is achieved, and the semiconductor refrigerating sheet is a core part of refrigeration. That is, if the temperature of the condensed water is set at 10 ℃, and the temperature of the upper end of the semiconductor chilling plate 14 is-50 ℃ after the chilling conversion by the semiconductor chilling plate 14, the experimental requirement can be satisfied.
In the embodiment, two refrigeration units are connected in series, and each refrigeration unit comprises two semiconductor refrigeration pieces 14 and two water-cooling plates 10.
The refrigeration unit is embedded into the bottom plate 9, a bottom plate clamping groove is formed in the bottom plate 9, the silicone tube 4 for carrying circulating condensate water and the electric wire 5 connected with the semiconductor refrigeration piece are both arranged in the designed bottom plate clamping groove, and therefore good protection can be achieved. The bottom plate 9 is designed after considering the sizes of the TwoVol2 denudation pool, the internal semiconductor chilling plate 14 and the water cooling plate 10, and the semiconductor chilling plate 14 and the water cooling plate 10 can be well placed in the bottom plate for fixation.
The front plates comprise three front plates, namely a first front plate 6, a second front plate 7 and a third front plate 8, wherein the three front plates are made of aluminum and mainly play a role in fixing the laser ablation frozen sample table.
The top plate 11 plays a role in supporting and refrigerating the biological sample, and the top plate 11 is made of polytetrafluoroethylene material and plays a role in heat preservation and cold insulation;
the carrier plate 15 is made of copper, and the thermal conductivity of copper is 401K (W/m.K), so that the carrier plate has excellent heat transfer capability.
The top plate 11 is provided with a carrying supporting plate through hole for matching with a carrying supporting plate 15, the carrying supporting plate 15 is provided with heat conduction mud 13, and the center of the heat conduction mud is provided with a groove for placing the glass slide 12.
The refrigeration unit is a water cooling plate and a semiconductor refrigeration plate which are adhered together by using heat-conducting silicon adhesive, is a core part of the whole refrigeration sample table and plays a refrigeration effect.
The bottom plate plays the fixed action to refrigerating unit, and the material of bottom plate is polytetrafluoroethylene equally for polytetrafluoroethylene has better heat preservation effect, makes the refrigeration effect of semiconductor refrigeration piece more stable.
Fig. 1 is an overall view of a laser ablation frozen sample stage, and two holes are formed in the middle, namely a pipeline inlet 16 and a pipeline outlet 17, which are mainly used for water and electric circuits.
Before the device is used, the laser ablation frozen sample table is sealed by using a sealant so as to be isolated from the outside, and the sealing of an ablation pool is ensured.
The dimensions of the various components in the laser ablation frozen sample stage are as follows:
the total length of the whole laser ablation frozen sample stage is 194.5mm, and the total width is 155.5 mm.
The material of roof 11 is polytetrafluoroethylene, and the length of roof 11 is 181mm, and the width is 155.5mm, and highly is 14mm, and the internal piping of 12 x 9mm and the wide recess of placing the refrigeration unit of 41mm have been designed to inside, and the surface of roof 11 has designed four through-holes that do not contain the interior hexagonal cylinder head screw class GB M5 of screw thread, and the side has designed two GB M5's screw hole.
The carrier plate 15 is of red copper and has overall dimensions of 155mm x 41mm x 2mm with four 2mm rounded corners. The size of the carrying supporting plate 15 can be tightly jointed and combined with the top plate 11, so that the improvement of the freezing effect is ensured. The carrying supporting plate 15 is provided with two cylindrical counter bores of GB M5 hexagon head bolts.
The length of bottom plate 9 is 181mm, 155.5mm, and the height is 14mm, and inside tunnel size and position are unanimous with roof 11, and the main purpose is that for the complete placement of refrigeration unit and the normal route of silicone tube 4 and electric wire 5, bottom plate 9 has six GB M5's screw hole altogether, and the GB M5 screw hole of bottom plate 9 and the through-hole of roof 11 and the counter bore of year layer board 15 are mutually aligned, use the hexagon socket head cap screw of GB M5 to closely link year layer board 15 and bottom plate 9 with roof 11, and there are two GB M4's screw hole in the side of bottom plate 9.
The first front plate 6 is an aluminum plate of 155.5mm × 36mm × 6.5mm as a whole, and has two hexagon head bolt through holes on its surface, four diameter holes of 5.3mm for screw connection, and a drawing cut of 85mm × 20mm in the middle.
The second front plate 7 is an aluminum plate having a whole size of 120.5mm × 32.5mm × 3mm, and has four holes having a diameter of 5.3mm on the surface, which are symmetrical with each other in the left-right direction and the up-down direction, for screw connection, and has a center cut by stretching 85mm × 20 mm.
The third front plate 8 is an aluminum plate having an overall size of 114mm × 26.3mm × 4mm, and has four holes having a diameter of 5.3mm on the surface thereof, which are symmetrical with respect to the left and right, and are used for screw connection, and has a center cut by stretching of 85mm × 20 mm.
The freezing device for the biological sample degradation imaging at the low temperature is adopted, and a self-assembly detection system for the biological sample degradation imaging at the low temperature is formed by matching a laser degradation system and an inductively coupled plasma mass spectrometer, and is used for detecting animal tissues, and the method comprises the following steps:
step one, after assembling a laser ablation frozen sample table, sealing by adopting a sealant, opening a circulating condensed water tank, setting the temperature to be 10 ℃, and carrying out the next operation after the temperature is stable (more than 10min, and the temperature fluctuation is within +/-0.05 ℃).
And step two, connecting a power supply to enable the frozen sample stage to start working, reducing the temperature to-20 ℃ within 4min, and when the temperature of the frozen sample stage is stabilized by laser ablation for 15min, controlling the temperature to-26.0 ℃ (adopting an external temperature measuring device 1 to measure the temperature).
Immediately placing the glass slide with the animal tissue slices on a carrying pallet of a laser ablation frozen sample table to ensure that the temperature of the glass slide is stable;
the preparation method of the animal tissue slice comprises the following steps: taking a mouse as an example: after the administration of the drug, the mice were sacrificed, and the organs thereof were subjected to a cryo-sectioning treatment, and the organs were cut into 10 μm thin sections using a cryo-microtome, placed on glass slides, and immediately placed on a frozen sample stage for ablation imaging.
The cooling curve of the laser ablation frozen sample stage of the embodiment is shown in fig. 4(a), and the temperature curve of the laser ablation frozen sample stage is shown in fig. 4(b) within 10 hours, which illustrates that the laser ablation frozen sample stage of the present invention has short freezing time, can reduce the temperature to-20 ℃ within 4min, reduces the waiting time of the detection personnel, and obtains stable freezing temperature in about 15 min; the utility model can keep very stable temperature, and the average temperature of 1h-10h is (-26.0 +/-0.1) DEG C.
Placing the laser ablation frozen sample stage in a laser ablation tank, screwing the laser ablation frozen sample stage by using a bolt, and performing flushing operation to ensure that impurity gas in the ablation tank is flushed; after the flushing is finished, the online mode is adjusted, the air flow is adjusted to be 50mL/min, and the total air flow is 700 mL;
step five, setting laser ablation parameters and energy intensity (Fluence): 2.5J/cm2(ii) a Repetition frequency (Rep Rate): 50 Hz; spot size (spot size): 50 μm; scan Speed (Scan Speed): 100 μm/s; the number of line-scan lines (The number of lines) is 100;
and step six, setting parameters of related elements in the plasma mass spectrum, adjusting a scanning parameter (sweep) to be 1, adjusting dwell time (dwell time) to be 50ms, and selecting the elements as the detected elements to construct the detection method.
And seventhly, carrying out degradation detection to obtain an animal tissue degradation image under a freezing condition, and analyzing the distribution condition of each element in the biological tissue according to the image.
The freezing apparatus for imaging the biological sample degradation at a low temperature of this example was subjected to cost accounting at a price of 4700 yuan.
Example 2
A freezing device for biological sample ablation imaging at low temperature has a structure schematic diagram shown in figure 3, and comprises a laser ablation frozen sample stage, a circulating condensed water tank 3 and a power supply 2;
the laser ablation frozen sample stage is shown in a schematic structural view in figure 1 and an explosion view in figure 2 and comprises a front plate, a top plate 11, a bottom plate 9, a refrigeration unit, a carrying supporting plate 15 and heat conducting mud 13;
a top plate clamping groove is formed in the top plate 11, the top plate clamping groove is matched with a bottom plate clamping groove formed in the bottom plate 9, and a front plate is arranged at the front end of the top plate to form a cooling cavity;
a fixed refrigeration unit clamping groove and a pipeline placing guide groove are also arranged on the bottom plate 9, and a pipeline inlet 17 and a pipeline outlet 16 are arranged on the front plate;
the refrigerating unit comprises a semiconductor refrigerating sheet 14 and a water cooling plate 10; the semiconductor refrigerating sheet 14 and the water cooling plate 10 are oppositely arranged and tightly connected; one side provided with the water cooling plate 10 is arranged on the fixed refrigeration unit clamping groove;
a carrying support plate through hole is further formed in the top plate 11, a carrying support plate 15 is arranged above the semiconductor refrigerating sheet 14 through the carrying support plate through hole, heat conduction mud 13 is coated above the carrying support plate 15, and a groove is formed in the middle of the heat conduction mud and used for placing a glass slide 12 of a biological sample;
the semiconductor refrigerating sheet 14 is connected with the power supply 2 through a wire 5; the water cooling plate 10 is communicated with the circulating condensation water tank 3 through a silicone tube 4.
The freezing device for the biological sample degradation imaging at the low temperature is adopted, and a self-assembly detection system for the biological sample degradation imaging at the low temperature is formed by matching a laser degradation system and an inductively coupled plasma mass spectrometer, and is used for detecting animal tissues, and the method comprises the following steps:
step one, after assembling a laser ablation frozen sample table, sealing by adopting a sealant, opening a circulating condensed water tank, setting the temperature to be 10 ℃, and carrying out the next operation after the temperature is stable (more than 10min, and the temperature fluctuation is within +/-0.05 ℃).
And step two, connecting a power supply to enable the frozen sample stage to start working, wherein the temperature can be reduced to-20 ℃ within 4min, and the temperature of the frozen sample stage is stabilized after 15min laser ablation, and is-26.0 ℃ when the temperature is stabilized (the temperature is measured by adopting an external temperature measuring device).
Immediately placing the glass slide with the plant tissue slices on a carrying pallet of a laser ablation frozen sample table to enable the temperature of the glass slide to be stable;
the preparation method of the plant tissue slice comprises the following steps: taking cucumber plants as an example: the method comprises the steps of selecting strong cucumber tissues to culture exogenous elements, slicing the cucumber tissues after several days, obtaining plant tissue slices on a freezing microtome, and immediately placing the plant tissue slices on a glass slide.
Placing the laser ablation frozen sample stage in a laser ablation tank, screwing the laser ablation frozen sample stage by using a bolt, and performing flushing operation to ensure that impurity gas in the ablation tank is flushed; after the flushing is finished, adjusting an online mode, wherein the air flow is adjusted to be 50mL/min, and the total air flow is 700 mL;
step five, setting laser ablation parameters and energy intensity (Fluence): 5J/cm2(ii) a Repetition frequency (Rep Rate): 50 Hz; spot size (spot size): 50 μm; scan Speed (Scan Speed): 100 μm/s; the number of line scanning lines (The number of lines) is 100;
and step six, setting parameters of related elements in the plasma mass spectrum, adjusting a scanning parameter (sweep) to be 1, adjusting dwell time (dwell time) to be 50ms, and selecting the elements as the detected elements to construct the detection method.
And seventhly, carrying out degradation detection to obtain a plant tissue degradation image under a freezing condition, and analyzing the distribution condition of each element in the biological tissue according to the image.
Example 3
A freezing device for imaging of biological sample degradation at low temperature, as in example 2.
The freezing device for biological sample ablation imaging at low temperature is adopted, and a self-assembly laser ablation frozen sample stage system is formed by matching a laser ablation pool and a plasma mass spectrum, so that single cells are detected, and the method comprises the following steps:
step one, after assembling a laser ablation frozen sample table, sealing by adopting a sealant, opening a circulating condensed water tank, setting the temperature to be 10 ℃, and carrying out the next operation after the temperature is stable (more than 10min, and the temperature fluctuation is within +/-0.05 ℃).
And step two, connecting a power supply to enable the frozen sample stage to start working, wherein the temperature can be reduced to-20 ℃ within 4min, and the temperature of the frozen sample stage is stabilized after 15min laser ablation, and is-26.0 ℃ when the temperature is stabilized (the temperature is measured by adopting an external temperature measuring device).
Immediately putting the array plate with the single cells on a carrying pallet of a laser ablation frozen sample table to stabilize the temperature;
the preparation method of the single cell array comprises the following steps: cell suspension was diluted to 10 using phosphate buffer solution6cells/mL, using a hemocytometerCells in suspension were counted for validation. Injecting into the array plate to make one cell enter one tunnel, and then using phosphate buffer solution to wash the upper end of the array plate lightly to wash away the redundant cells. Immediately freezing by using liquid nitrogen, and quickly placing on a frozen sample table;
placing the laser ablation frozen sample stage in a laser ablation tank, screwing the laser ablation frozen sample stage by using a bolt, and performing flushing operation to ensure that impurity gas in the ablation tank is flushed; after the flushing is finished, the online mode is adjusted, the air flow is adjusted to be 50mL/min, and the total air flow is 700 mL;
step five, setting laser ablation parameters and energy intensity (Fluence): 5J/cm2(ii) a Repetition frequency (Rep Rate): 50 Hz; spot size (spot size): 50 μm; the number of line-scan lines (The number of circles) is 500;
and step six, setting parameters of related elements in the plasma mass spectrum, adjusting a scanning parameter (sweep) to be 1, adjusting dwell time (dwell time) to be 50ms, and selecting the elements as the detected elements to construct the detection method.
And seventhly, carrying out degradation detection, namely obtaining single cell degradation detection under a freezing condition, and analyzing the distribution condition of each element in the biological tissue according to imaging.
Comparative example
A freezing device for use in imaging of biological sample degradation at low temperature, as in example 1, except that:
the center of the laser ablation normal temperature sample stage is provided with a light hole, light is directly irradiated to the biological sample from the lower part, the visual field can be clearer and brighter, and therefore accurate focusing of the biological sample is achieved, and the sample stage is shown in fig. 5 (a). The embodiment of the utility model provides an in 1 not set up the light trap, the function of focus relies on heat conduction mud to realize, through the strong reflectance ability of heat conduction mud to the light, the field of vision is brighter in making the degradation pond to obtain accurate focusing effect, use heat conduction mud simultaneously also can obtain more even refrigeration effect, its field of vision focusing effect picture is seen in figure 5 (b).
Claims (9)
1. A freezing device for biological sample degradation imaging at low temperature is characterized by comprising a laser degradation frozen sample stage, a circulating condensed water tank and a power supply;
the laser ablation frozen sample stage comprises a front plate, a top plate, a bottom plate, a refrigeration unit, a carrying supporting plate and heat conducting mud;
a top plate clamping groove is formed in the top plate, the top plate clamping groove is matched with a bottom plate clamping groove formed in the bottom plate, and a front plate is arranged at the front end of the top plate to form a cooling and heat dissipation cavity;
the refrigerating unit comprises a semiconductor refrigerating sheet and a water-cooling plate; the semiconductor refrigerating plate and the water cooling plate are arranged oppositely and tightly connected; one side of a water cooling plate in the refrigeration unit is arranged on the bottom plate;
a carrying support plate through hole is further formed in the top plate, the carrying support plate is arranged above the semiconductor refrigerating sheet through the carrying support plate through hole, heat conduction mud is coated on the carrying support plate, and a groove is formed in the middle of the heat conduction mud and used for placing a glass slide of a biological sample;
the semiconductor refrigerating sheet is connected with a power supply through a wire; the water cooling plate is communicated with the circulating condensed water tank through a pipeline.
2. A freezing apparatus for biological sample degradation imaging at low temperatures according to claim 1 wherein the refrigeration unit is a plurality of series connected.
3. The freezing apparatus for biological sample degradation imaging at low temperature according to claim 1, wherein a heat conductive silica gel tight connection is arranged between the conductor refrigeration sheet and the water cooling plate.
4. The freezing apparatus for biological sample degradation imaging at low temperature according to claim 1, wherein the semiconductor refrigeration sheet and the carrier plate are connected by heat-conducting silica gel for uniform conduction of refrigeration effect.
5. A freezer for imaging degradation of biological samples at cryogenic temperatures as recited in claim 1, wherein the floor is sized according to the size of the refrigeration unit and the size of the degradation bath.
6. A freezing apparatus for biological sample degradation imaging at low temperature as claimed in claim 1, wherein a groove for fixing the refrigeration unit and a channel for placing the pipeline are further provided on the bottom plate.
7. A freezer for imaging degradation of biological samples at cryogenic temperatures as recited in claim 1, wherein the top and bottom plates cooperate to form a line inlet and a line outlet.
8. The freezing apparatus for ablation imaging of biological samples at low temperature according to claim 1, wherein the front plate comprises a first front plate, a second front plate and a third front plate, and the front plates are made of aluminum and used for fixing the laser ablation frozen sample stage.
9. A detection system for imaging degradation of a biological sample at a low temperature is characterized by comprising the refrigerating device for imaging degradation of the biological sample at the low temperature as claimed in any one of claims 1 to 8, and further comprising a temperature measuring device, a laser degradation system and an inductively coupled plasma mass spectrometer;
the temperature measuring device is used for measuring the temperature of the refrigerating device for the denudation imaging of the biological sample at low temperature;
the laser ablation system is used for ablating the biological sample on the glass slide;
and the inductively coupled plasma mass spectrometer is used for performing mass spectrometry and data processing on the denuded sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117153660A (en) * | 2023-11-01 | 2023-12-01 | 中国地质大学（武汉） | Automatic sampling solution laser ablation pond |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117153660A (en) * | 2023-11-01 | 2023-12-01 | 中国地质大学（武汉） | Automatic sampling solution laser ablation pond |
| CN117153660B (en) * | 2023-11-01 | 2024-01-12 | 中国地质大学（武汉） | Automatic sampling solution laser ablation pond |
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