CN114088507B - High-intensity focused ultrasound dewaxing system and dewaxing device for paraffin embedded tissues - Google Patents
High-intensity focused ultrasound dewaxing system and dewaxing device for paraffin embedded tissues Download PDFInfo
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Abstract
The invention provides a high-intensity focused ultrasound dewaxing system for paraffin embedded tissues, which comprises a singlechip, a signal source circuit, a power supply, a power amplifying circuit and a focusing piezoelectric transducer, wherein the singlechip is connected with the signal source circuit; the signal source circuit outputs two paths of square wave signals which are respectively output from an OUTA end and an OUTB end of the signal source circuit after PWM modulation; the power amplifying circuit is used for carrying out voltage gating and amplifying on signals output by the OUTA end and the OUTB end, outputting the signals to the focusing piezoelectric transducer, and driving the focusing piezoelectric transducer to generate PWM modulated ultrasonic excitation so as to enable liquid in the sample tube to vibrate, so that paraffin is crushed and emulsified. The dewaxing system provides high-intensity focused sound wave pulse, can crush and emulsify paraffin in a focusing area, realizes thorough dewaxing through a physical method, replaces the traditional dewaxing scheme using toxic xylene solvents, is environment-friendly and nontoxic, improves the dewaxing effect and shortens the operation time.
Description
Technical Field
The invention relates to the technical field of high-intensity focused ultrasound, in particular to a high-intensity focused ultrasound dewaxing system and a dewaxing device for paraffin embedded tissues.
Background
Many research objects in modern molecular biology are tissues, and after the tissues leave the organism, the tissues die and generate tissue putrefaction, and the original normal structure of the tissues is lost, so the tissues are firstly subjected to the steps of fixing, paraffin embedding, slicing, staining and the like to avoid death of the cell tissues, so that the morphological structure of the tissues can be clearly identified later.
Paraffin embedded FFPE (format in-fixed Paraffin-emmedding) is a sample preparation technique for fixing a tissue sample by formalin and performing Paraffin embedding, and is widely applied to the fields of pathology detection, forensic identification, clinical research and the like. However, extracting nucleic acid from FFPE samples is challenging because DNA and RNA in FFPE samples have been partially degraded and DNA has been tightly cross-linked with histones, and paraffin wax inhibits the penetration effect of the lysate on tissue, thus requiring thorough dewaxing.
The traditional FFPE chemical dewaxing method needs organic solvents such as dimethylbenzene and mineral oil, is difficult to process samples with thicker slices or long storage time, has incomplete dewaxing effect, is toxic to human bodies, and is easy to interfere with the application on the subsequent molecular level.
Disclosure of Invention
In order to achieve the above object, the present invention is achieved by the following technical solutions.
The invention provides a high-intensity focused ultrasound dewaxing system for paraffin embedded tissues, which comprises a singlechip, a signal source circuit, a power supply, a power amplifying circuit and a focusing piezoelectric transducer, wherein the singlechip is connected with the signal source circuit; the output end of the singlechip is respectively connected with the signal source circuit and the power input end; the signal source circuit and the output end of the power supply are respectively connected with the input end of the power amplifying circuit; the output end of the power amplification circuit is connected with the input end of the focusing piezoelectric transducer; wherein,
the singlechip controls the two paths of square wave signals which are opposite in output level of the signal source circuit and have the frequency equal to the resonant frequency of the focusing piezoelectric transducer, and the square wave signals are respectively output from an OUTA end and an OUTB end of the signal source circuit after PWM modulation of the singlechip 12;
the power amplifying circuit is used for carrying out voltage gating and amplifying on signals output by the OUTA end and the OUTB end, outputting the signals to the focusing piezoelectric transducer, and driving the focusing piezoelectric transducer to generate PWM modulated ultrasonic excitation so as to enable liquid in a sample tube positioned in a focusing area to vibrate, and enabling paraffin in the sample tube to be broken and emulsified.
Preferably, the power amplifying circuit is a class D power amplifying circuit.
Preferably, the power amplifying circuit comprises a first high-speed optocoupler, a second high-speed optocoupler, a grid driver, a current limiting resistor R1, a current limiting resistor R2, an NMOS tube Q1, an NMOS tube Q2, a radio frequency choke RFC and a high-frequency transformer T1;
the OUTA end and the OUTB end of the signal source circuit are respectively connected with the first high-speed optical coupler and the second high-speed optical coupler; the first high-speed optocoupler output end and the second high-speed optocoupler output end are respectively connected with two input ends of the grid driver; the OUTA end of the grid driver is sequentially connected with the current limiting resistor R1 and the NMOS tube Q1; the OUTB end of the grid driver is sequentially connected with the current limiting resistor R2 and the NMOS tube Q2;
the power supply is a programmable digital voltage source, and the power supply is respectively connected to the circuits of the NMOS tube Q1 and the NMOS tube Q2 after sequentially passing through the radio frequency choke coil RFC and the high-frequency transformer T1.
Preferably, the system further comprises an upper computer which is electrically connected with the singlechip and used for receiving an operation instruction and sending the instruction to the singlechip.
Preferably, the power amplifier further comprises an impedance matching structure connected to the output end of the power amplifier circuit; the impedance matching structure is connected with the focusing piezoelectric transducer and is used for matching the impedance of the focusing piezoelectric transducer.
Preferably, the signal source circuit includes:
the direct digital frequency synthesis module is used for outputting two paths of square wave signals which are opposite in level and have the frequency equal to the resonance frequency of the focusing piezoelectric transducer;
and the two digital AND gates are used for performing AND operation on the square wave signal and the PWM wave signal.
Preferably, the resonance frequency of the focusing piezoelectric transducer is 100 kHz-10 MHz.
The invention also provides a dewaxing device comprising a high intensity focused ultrasound dewaxing system for paraffin embedded tissue.
Preferably, the container is provided with a containing cavity and a fixing frame; the cavity is filled with an ultrasonic transmission medium; the fixing frame is used for fixing the sample tube;
the focusing piezoelectric transducer is fixed in the accommodating cavity; the sample tube is located within a focal region of the focused piezoelectric transducer.
Preferably, the focusing piezoelectric transducer is fixed at the bottom of the cavity.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a high-intensity focused ultrasonic dewaxing system for paraffin embedded tissues, which provides high-intensity focused sonic pulses, can crush and emulsify paraffin in a focusing area, realizes thorough dewaxing by a physical method, replaces the traditional dewaxing scheme of using toxic xylene solvents, is environment-friendly and nontoxic, improves the dewaxing effect and shortens the operation time.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the technical means of the present invention, and is to be implemented in accordance with the contents of the specification, as follows, in accordance with the preferred embodiments of the present invention, as hereinafter described in detail with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail by the following examples and the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a schematic diagram of a dewaxing system according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a circuit for connecting a singlechip with a signal source circuit;
FIG. 3 is a schematic diagram of a power amplifier circuit according to the present invention;
fig. 4 is a schematic structural view of the device body of the present invention.
In the figure:
10. a dewaxing system; 11. an upper computer; 12. a single chip microcomputer; 13. a signal source circuit; 131. a direct digital frequency synthesis module; 14. a power supply; 15. a power amplifying circuit; 151. a first high-speed optocoupler; 152. a second high-speed optocoupler; 153. a gate driver; 16. an impedance matching structure;
20. a device body; 21. a container; 211. a cavity; 212. a fixing frame; 22. elastic sealant;
30. a sample tube; 31. embedding the tissue in paraffin;
40. a focal region.
Detailed Description
The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses a device for practicing the invention. In the drawings, the shape and size may be exaggerated for clarity, and the same reference numerals will be used throughout the drawings to designate the same or similar components. In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, etc. are based on the orientation or positional relationship shown in the drawings. In particular, "height" corresponds to the top-to-bottom dimension, "width" corresponds to the left-to-right dimension, and "depth" corresponds to the front-to-back dimension. These relative terms are for convenience of description and are not generally intended to require a particular orientation. Terms (e.g., "connected" and "attached") referring to an attachment, coupling, etc., refer to a relationship wherein these structures are directly or indirectly secured or attached to one another through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.
Example 1
The invention provides a high-intensity focused ultrasound dewaxing system for paraffin embedded tissues, which is shown in fig. 1, and comprises a dewaxing system 10, wherein the dewaxing system 10 comprises a singlechip 12, a signal source circuit 13, a power supply 14, a power amplifying circuit 15 and a focusing piezoelectric transducer 17; the output end of the singlechip 12 is respectively connected with the input ends of the signal source circuit 13 and the power supply 14; the output ends of the signal source circuit 13 and the power supply 14 are respectively connected with the input end of the power amplifying circuit 15; the output end of the power amplification circuit 15 is connected with the input end of the focusing piezoelectric transducer 17; wherein,
the singlechip 12 generates a PWM wave signal; the singlechip 12 controls the signal source circuit 13 to output two paths of square wave signals with opposite levels and frequency equal to the resonant frequency of the focusing piezoelectric transducer 17, and the square wave signals are respectively output from the OUTA end and the OUTB end of the signal source circuit 13 after PWM modulation of the singlechip 12;
the power amplifying circuit 15 is configured to perform voltage gating and amplifying on the signals output from the OUTA end and the OUTB end, switch and output two amplified voltage signals to the focusing piezoelectric transducer 17, and drive the focusing piezoelectric transducer 17 to generate PWM modulated ultrasonic excitation, so that the liquid in the sample tube 30 located in the focusing region oscillates, and the paraffin embedded outside the tissue in the sample tube 30 is broken and emulsified in the liquid. Specifically, the singlechip 12 is used as a control core of the dewaxing system and is used for controlling the signal source circuit 13 and adjusting the power of the circuit. The singlechip 12 generates a pulse width modulation PWN waveform with adjustable frequency and duty ratio, and is used for modulating the signal source circuit 13 to output a positive phase square wave signal and a negative phase square wave signal; i.e. the effective power of the focused ultrasound is adjusted by PWN modulation. The power amplification circuit continuously switches and amplifies two paths of square wave signals modulated by the gating PWN, so that the focusing piezoelectric transducer 17 generates ultrasonic excitation with different powers, ultrasonic waves act on a lysis solution which is contained in the sample tube 30 and is used for soaking paraffin embedded tissues through a transmission medium, the lysis solution oscillates to enable paraffin embedded outside the tissues in the sample tube 30 to be broken into tiny particles, and the tiny paraffin particles are emulsified in an aqueous medium in the sample tube 30, so that dewaxing is realized. Dewaxing system 10 provides high intensity focused sonic pulses capable of breaking and emulsifying paraffin in the focal zone to physically achieve thorough dewaxing, replaces conventional dewaxing schemes using toxic xylene solvents, is environmentally friendly and nontoxic, improves dewaxing efficiency, and shortens operating time.
In an embodiment, the power amplifying circuit 15 is a class D power amplifying circuit, which provides high power efficiency and can be used for driving a high-power circuit.
Further, as shown in fig. 3, the power amplifying circuit 15 includes a first high-speed optocoupler 151, a second high-speed optocoupler 152, a gate driver 153, a current limiting resistor R1, a current limiting resistor R2, an NMOS transistor Q1, an NMOS transistor Q2, a radio frequency choke RFC, and a high-frequency transformer T1;
the OUTA end and the OUTB end of the signal source circuit 13 are respectively connected with the INA end of the first high-speed optocoupler 151 and the INB end of the second high-speed optocoupler 152; the output end of the first high-speed optocoupler 151 and the output end of the second high-speed optocoupler 152 are respectively connected with two input ends of the gate driver 153, that is, the output end of the first high-speed optocoupler 151 is connected with the INA end of the gate driver 153, and the output end of the second high-speed optocoupler 152 is connected with the INB end of the gate driver 153; the OUTA end of the gate driver 153 is sequentially connected with the current limiting resistor R1 and the NMOS transistor Q1; the OUTB end of the gate driver 153 is sequentially connected with the current limiting resistor R2 and the NMOS tube Q2;
the power supply 14 is a programmable digital voltage source, and is connected to the circuits of the NMOS transistor Q1 and the NMOS transistor Q2 after passing through the radio frequency choke RFC and the high frequency transformer T1 in sequence.
Specifically, a programmable digital voltage source is used as a power supply of the power amplifying circuit, and the singlechip 12 controls the programmable digital voltage source to generate adjustable voltage for adjusting instantaneous output power; in a specific embodiment, the model of the programmable digital voltage source is DKP6012, and the singlechip 12 controls the DKP6012 to generate 0-20V adjustable voltage. The first high-speed optocoupler 151 and the second high-speed optocoupler 152 are respectively used for transmitting signals output by the OUTA end and the OUTB end of the signal source circuit 13 and isolating the signal source circuit 13. The gate driver 153 provides high current driving capability, reduces the on and off time of the NMOS transistors Q1 and Q2, improves the frequency characteristics of the circuit, and reduces the switching loss. The current limiting resistor R1 is used for preventing the gate driving current of the NMOS transistor Q1 from being too large, and the current limiting resistor R2 is used for preventing the gate driving current of the NMOS transistor Q2 from being too large. The radio frequency choke RFC is used for suppressing current variation, is matched with the power supply 14, and is equivalent to a constant current source, if the radio frequency choke RFC is not adopted, the current fluctuation required by the power amplification part is large and the frequency is high, and the DC-DC power supply cannot meet the requirement. The high-frequency transformer T1 is wound by a nickel-zinc ferrite core and an enamelled wire, and has good high-frequency characteristics. Further, the first high-speed optocoupler 151 and the second high-speed optocoupler 152 are 6N137 optocouplers. Further, the gate driver 153 is an MC33152 driver.
The dewaxing system adopts a programmable digital voltage source and a PWN modulation mode to respectively adjust the instantaneous power and the effective power of the focused ultrasound, generates ultrasonic excitation of different powers, and has high flexibility. The prior switch type power amplifying circuit adopts more H-bridge circuits or D-type power amplifiers with positive and negative power supplies. The H bridge circuit needs 4 MOSFETs and corresponding driving circuits, and is relatively complex in structure; the class D power amplifying circuit with positive and negative power supplies needs to provide high-power positive and negative power supplies, and PMOS elements with high price, high on-resistance and low speed are used; meanwhile, the two circuits have no voltage amplification effect, and the amplitude of the power supply is required to be larger. The power amplifying circuit 15 in the scheme adopts two NMOS tubes and a high-frequency transformer T1, has a simple circuit structure, can amplify current and voltage simultaneously, adopts a piezoelectric transducer with 2MHz, a band cable and measured impedance of (0.858+j3.168) omega, diameter of 38mm and curvature radius of 38mm for verification, and can obtain the circuit efficiency of 87.35 percent through TINA9 software simulation. In addition, the power amplification circuit 15 is small in size, simple in structure and low in cost.
The power amplifying circuit 15 has three operating states according to the output signal conditions of the OUTA terminal and the OUTB terminal of the signal source circuit 13:
(1) The NMOS tube Q1 and the NMOS tube Q2 are closed, and the power amplifying circuit 15 has no voltage output;
(2) The NMOS tube Q1 is conducted, the NMOS tube Q2 is closed, current flows through the high-frequency transformer T1 and the NMOS tube Q1 from the radio frequency choke RFC, and finally the current reaches the ground, and reverse voltage is generated at the output end of the high-frequency transformer T1;
(3) The NMOS tube Q1 is closed, the NMOS tube Q2 is conducted, current flows through the high-frequency transformer T1 and the NMOS tube Q2 from RFC, finally, the current reaches the ground, and the output end of the high-frequency transformer T1 generates forward voltage.
For PWM modulated signals, during low levels, the circuit is in a first state; during the high level, the circuit switches back and forth between the second and third states, so that the input terminal of the high frequency transformer T1 generates current drive and outputs to the focusing piezoelectric transducer 17.
Further, the model of the NMOS tube Q1 and the model of the NMOS tube Q2 are IRFB4020.
In an embodiment, as shown in fig. 1, the device further includes an upper computer 11 electrically connected to the single-chip microcomputer 12, and configured to receive an operation instruction and send the instruction to the single-chip microcomputer 12. Specifically, the user can set experimental parameters and procedures through the operation interface of the upper computer 11 to perform dewaxing work of different experiments. Further, the upper computer 11 is electrically connected with the singlechip 12 through a USB cable.
In one embodiment, as shown in fig. 1, the power amplifier circuit further comprises an impedance matching structure 16, which is connected to the output end of the power amplifier circuit 15; the impedance matching structure 16 is connected to the focusing piezoelectric transducer 17 for matching the impedance of the focusing piezoelectric transducer 17. Specifically, the piezoelectric ceramic of the focusing piezoelectric transducer 17 exhibits capacitance at the resonance frequency, and the direct drive effective power is small and the harmonics are large. The impedance matching structure 16 makes the load of the focusing piezoelectric transducer 17 purely resistive. It is necessary to measure the impedance of the focusing piezoelectric transducer 17 and perform impedance matching based on the schmitt diagram to make it a purely resistive load.
In one embodiment, as shown in fig. 2, the signal source circuit 13 includes:
the direct digital frequency synthesis module 131 is configured to output two square wave signals with opposite levels and a frequency equal to the resonant frequency of the focusing piezoelectric transducer 17;
and the two digital AND gates are used for performing AND operation on the square wave signal and the PWM wave signal.
The power supply 14 is a programmable digital power supply. Specifically, the SPI communication port of the singlechip 12 is connected to the SPI communication port of the direct digital frequency synthesis module 131, so as to control the signal source circuit 13 to output two paths of square wave signals with opposite levels, and the two paths of square wave signals with opposite levels are respectively transmitted to two digital and gate circuits. The PWN port of the singlechip 9 is connected to the two digital and gate circuits, respectively, so as to transmit PWM wave signals to the two digital and gate circuits, respectively. In the figure, "&" indicates a digital AND gate. In one embodiment, the direct digital frequency synthesis module 131 is an AD9850 DDS chip.
In one embodiment, the resonant frequency of the focusing piezoelectric transducer 17 is 100 kHz-10 MHz, and the focusing range is controlled to concentrate ultrasonic energy.
Example 2
The present invention provides a dewaxing device, as shown in figures 1 to 4, comprising a device body 20, the device body 20 comprising a high intensity focused ultrasound dewaxing system for paraffin embedded tissue as described above. The dewaxing system is adopted to replace the use of toxic xylene solvent for dewaxing, is safe and nontoxic, improves the dewaxing effect and shortens the operation time.
In one embodiment, the device body 20 includes a container 21, which is provided with a cavity 211 and a fixing frame 212; the accommodating cavity 211 is filled with an ultrasonic transmission medium; the fixing frame 212 is used for fixing the sample tube 30;
the focusing piezoelectric transducer 17 is fixed in the accommodating cavity 211; the sample tube 30 is located within a focal region 40 of the focusing piezoelectric transducer 17. Specifically, paraffin-embedded tissue 31 is placed within sample tube 30 and a lysing solution is added to soak the paraffin-embedded tissue. Other electronic components of the dewaxing system are integrated on a circuit board (not shown in the figure), and when the circuit is in operation, the focusing piezoelectric transducer 17 converts electric energy into mechanical energy, generates ultrasonic waves with different frequencies, propagates to the sample tube 30 under the propagation of a transmission medium, generates high-pressure sonic pulses, and enables the lysate in the sample tube 30 to vibrate, so that paraffin in the paraffin embedding tissue 31 in the sample tube 30 is broken and emulsified in the water of the lysate. Simple structure and easy operation, and high wax breaking efficiency.
Further, the transmission medium in the chamber 211 is ultrapure water.
In one embodiment, the focusing piezoelectric transducer 17 is fixed at the bottom of the cavity 211, and the fixing frame 212 is located above the focusing piezoelectric transducer 17; the sample tube 30 is vertically fixed to the fixing frame 212.
In an embodiment, the focusing piezoelectric transducer 17 is fixed on the surface wall of the cavity 211 by using an elastic sealant 22, and the focusing piezoelectric transducer 17 leads out two electrode wires to realize circuit connection.
Example 3
Dewaxing effect verification of dewaxing system 10 includes the steps of:
1. tissue sample embedding and manufacturing
3 female mice of SPF grade Balb/c are purchased from Yi Si laboratory animal technology Co., vinca, weight is 16+ -2 g, age is 6 weeks, the mice are killed by adopting a vertebra dislocation method, the four limbs of the mice are fixed on a foam plate by using a scalpel and surgical scissors, fresh double-lung and leg musculature blocks (with the thickness not more than 0.5 cm) of the mice are picked up, surface blood stains are removed by brushing twice in phosphate buffer solution, the mice are put into pre-purchased general tissue fixing solution (10% formalin, kansael medical examination Co., ltd.) and the tissue blocks are fully immersed by the fixing solution, soaked for 24 hours at 4 ℃, so that the proteins of tissues and cells are denatured and solidified, and the original morphological structure of the cells is maintained. Gradually removing water from tissue blocks by using low-concentration to high-concentration alcohol as a dehydrating agent, soaking the tissue blocks in xylene, and replacing the alcohol in the tissue blocks with the xylene.
In one embodiment, the alcohol dehydration is performed as follows:
(1) After rinsing with ultrapure water, placing the tissue block into a 5mL centrifuge tube;
(2) Dehydrating with 50% ethanol for 1.5h;
(3) Dehydrating with 70% ethanol for 1.5h;
(4) Dehydrating with 85% ethanol for 1 hr;
(5) Dehydrating with 95% ethanol for 0.5h;
(6) Dehydrating the absolute ethyl alcohol for 0.5h, and repeating the dehydration for one time;
(7) The xylene soak was repeated once for 0.5 h.
Placing the transparent tissue block into melted paraffin, and placing into a paraffin dissolving box for heat preservation. Embedding after paraffin is completely immersed into the tissue block, wherein the specific operation is as follows: pouring the melted paraffin into a container prepared in advance, rapidly clamping a tissue block soaked with the paraffin, placing the tissue block into the container, placing the container in a position, cooling the container on a cooling table of a paraffin embedding machine, and obtaining paraffin embedded tissue after the paraffin embedded tissue block is solidified into a block.
2. Wax breaking by using device body 20
(1) Paraffin-embedded tissue sections (total thickness. Ltoreq.40 μm) 5-10 μm thick were placed in a sample tube 30 containing a lysate consisting of 100mM Tris-HCl pH 8.0, 200mM NaCl,10mM EDTA pH 8.0 and 1.0% SDS;
(2) Placing the sample tube 30 within the focal region of the piezoelectric ceramic of the dewaxing system 10;
(3) Starting ultrasonic treatment and paraffin emulsification;
(4) Adding proteinase K and incubating for 20min at 56 ℃;
(5) Centrifuging at 8000rpm for 5 minutes;
(6) The supernatant was used for RNA extraction and the precipitate was used for DNA extraction.
3. Determination of DNA and RNA concentration
Several pieces of muscle tissue sample 1, muscle tissue sample 2, muscle sample tissue 3, lung tissue sample 1, lung tissue sample 2, and lung tissue sample 3 were prepared.
Taking 1 part of each of the muscle tissue sample 1, the muscle tissue sample 2, the muscle tissue sample 3, the lung tissue sample 1, the lung tissue sample 2 and the lung tissue sample 3, and performing a second treatment by adopting a dewaxing system 10 to obtain supernatant and precipitation liquid. The dewaxing system 10 comprises an upper computer 11, a singlechip 12, a signal source circuit 13, a power supply 14, a power amplifying circuit 15, an impedance matching structure 16 and a focusing piezoelectric transducer 17; the power amplifying circuit 15 includes a first high-speed optocoupler 151, a second high-speed optocoupler 152, a gate driver 153, a current limiting resistor R1, a current limiting resistor R2, an NMOS tube Q1, an NMOS tube Q2, a radio frequency choke RFC, and a high-frequency transformer T1; the power supply 14 is a programmable digital voltage source; the model of the NMOS tube Q1 and the model of the NMOS tube Q2 are IRFB4020; the signal source circuit 13 includes a direct digital frequency synthesis module 131, two digital and gates. The first high-speed optocoupler 151 and the second high-speed optocoupler 152 are 6N137 optocouplers; the gate driver 153 is an MC33152 driver. The singlechip 12 generates a PWM waveform with a frequency of 1kHz and a duty cycle of 25%. The resonance frequency of the focusing piezoelectric transducer 17 is 2MHz, and the power amplification circuit 15 switches and outputs 2MHz current drive in two states of NMOS transistor Q1 on, NMOS transistor Q2 off or NMOS transistor Q1 on and NMOS transistor Q2 off at the frequency of 2 MHz.
The concentration of DNA and RNA was determined separately using a Nanodrop spectrophotometer.
Comparative example 1
1 part of each of the muscle tissue sample 1, the muscle tissue sample 2, the muscle tissue sample 3, the lung tissue sample 1, the lung tissue sample 2 and the lung tissue sample 3 was dewaxed with xylene, and then the DNA concentration was measured.
The method specifically comprises the following steps:
(1) Cutting paraffin samples into slices with the thickness of 5-10 mu m, placing paraffin embedded tissue slices with the thickness of 5-10 mu m (the total thickness is less than or equal to 40 mu m) into a 1.5mL sterile centrifuge tube, adding 1mL of dimethylbenzene, and severely swirling for 10sec;
(2) Centrifuging at 12000rpm (-13400 Xg) at room temperature (15-25deg.C) for 2min, and removing supernatant with gun head, taking care not to suck precipitate;
(3) 1mL of ethanol (96-100%) was added to the precipitate, and vortexed for 10sec;
(4) Centrifuge at 12000rpm (13400 Xg) for 2min at room temperature, remove supernatant with a gun head, and carefully avoid sucking up sediment;
(5) Opening the tube cover, and standing at room temperature (15-25deg.C) or 37deg.C for 5-10min until residual ethanol is completely volatilized;
(6) Adding 200 mu L of buffer GA to resuspend the sediment, adding 20 mu L of protease K into the sediment, thoroughly vortex mixing, incubating for 1h at 56 ℃ (or until the sample has been completely lysed);
(7) Incubating for 1h at 90 ℃;
(8) To remove RNA, 2. Mu.L RNase A (100 mg/mL) was added and incubated at room temperature for 2min;
(9) 200 mu L of buffer GB is added and vortex mixed uniformly; then 250 mu L of ethanol (96-100%) is added and mixed by vortex; removing residual liquid drops on the cover and the side wall by simple centrifugation;
(10) Transferring the complete lysate to an adsorption column CR2, covering a cover, centrifuging at 8000rpm (about 6,000Xg) at room temperature for 2min, discarding the waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
(11) Adding 500 μl of buffer GD into the adsorption column CR2, covering the buffer GD, centrifuging at 8000rpm (6000 Xg) at room temperature for 60sec, discarding the waste liquid in the collection tube, and placing the adsorption column CR2 back into the collection tube;
(12) Adding 600 μl of rinse solution PW to the adsorption column CR2, covering the cover, centrifuging at 8000rpm (6000×g) at room temperature for 60sec, discarding the waste liquid in the collection tube, and placing the adsorption column CR2 back into the collection tube;
(13) Repeating step (12);
(14) Centrifuging at 12000rpm (13400 Xg) for 2min at room temperature, and discarding the waste liquid in the collecting pipe; placing the adsorption column cover at room temperature for 2-5min to thoroughly dry the residual rinsing liquid in the adsorption material;
(15) Transferring the adsorption column CR2 into a clean 1.5mL centrifuge tube, suspending and dripping 30-100p L eluting buffer TE or ddH2O preheated at 65 ℃ into the middle part of the adsorption film, standing for 2-5min at room temperature, centrifuging at 12000rpm (13400 Xg) for 2min, and collecting DNA;
(16) The DNA concentration was measured separately using a Nanodrop spectrophotometer.
Comparative example 2
1 part of each of the muscle tissue sample 1, the muscle tissue sample 2, the muscle tissue sample 3, the lung tissue sample 1, the lung tissue sample 2, and the lung tissue sample 3 was dewaxed with xylene, and then the RNA concentration was measured.
The method specifically comprises the following steps:
(1) Cutting paraffin samples into slices with the thickness of 5-10 mu m, placing paraffin embedded tissue slices with the thickness of 5-10 mu m (the total thickness is less than or equal to 40 mu m) into a 1.5mL sterile centrifuge tube, adding 1mL of dimethylbenzene, and severely swirling for 10sec;
(2) Centrifuging at 12000rpm (-13400 Xg) at room temperature (15-25deg.C) for 2min, and removing supernatant with gun head, taking care not to suck precipitate;
(3) Adding 1mL of absolute ethyl alcohol into the sediment, and uniformly mixing by vortex;
(4) Centrifuging at 12000rpm (-13400 Xg) for 2min at room temperature (15-25deg.C);
(5) The supernatant was aspirated with the gun head, taking care not to aspirate sediment (carefully aspirate residual ethanol with a new gun head);
(6) Opening the tube cover, and standing at room temperature (15-25deg.C) or 37deg.C for 10min until the residual ethanol is completely volatilized;
(7) 200. Mu.L of lysate RF and 10. Mu.L of protease K are added to the pellet, and thoroughly vortexed;
(8) Incubating at 55 ℃ for 15min and then incubating at 80 ℃ for 15min;
(9) Centrifuging at room temperature (15-25deg.C) at 12000rpm (-13400 Xg) for 5min, transferring supernatant into a new RNase-Free centrifuge tube;
(10) Adding 220 mu L of buffer RB, and uniformly mixing by vortex;
(11) 660. Mu.L of absolute ethanol is added, and the mixture is stirred and mixed uniformly (precipitation can occur);
(12) Transferring 700 μL of the solution and precipitating into an adsorption column CR3 (the adsorption column is placed in a collecting pipe), centrifuging at 12000rpm (13400 Xg) for 1min, discarding the waste liquid in the collecting pipe, and placing the adsorption column back into the collecting pipe;
(13) Repeating step 14 until all the solution and precipitate completely pass through the adsorption column CR3, discarding the waste liquid, and placing the adsorption column CR3 back into the collecting pipe;
(14) Preparing DNase I working solution: 10. Mu.L of DNase I stock was placed in a fresh RNase-Free centrifuge tube, 70. Mu.L of RDD buffer was added and gently mixed.
(15) Adding 80 mu L of DNase I working solution into the center of an adsorption column CR3, and standing at room temperature for 15min;
(16) Adding 500 μl deproteinized liquid RW1 into the adsorption column CR3, centrifuging at 12000rpm (13400 Xg) at room temperature (15-25deg.C) for 30-60sec, discarding the waste liquid, and placing the adsorption column back into the collection tube;
(17) Adding 500 μl of rinsing liquid RW (before use, checking whether ethanol has been added) into the adsorption column CR3, standing at room temperature for 2min, centrifuging at 12000rpm (13400×g) for 30-60sec, discarding the waste liquid, and placing the adsorption column CR3 back into the collection tube;
(18) Repeating step (19);
(19) Centrifuging at 12000rpm (-13400 Xg) for 2min at room temperature (15-25deg.C), and pouring out the waste liquid; placing the adsorption column CR3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
(20) Transferring the adsorption column CR3 into a new RNase-Free centrifuge tube, suspending and dripping 30-100 mu L of RNase-Free ddH20 into the middle part of the adsorption film, standing at room temperature for 2min, and centrifuging at 12000rpm (about 13400 Xg) for 2min to obtain an RNA solution;
(21) RNA concentration was determined using Nanodrop.
Example 4
Comparing the dewaxing results of dewaxing system 10 in example 3 with the dewaxing results of xylenes in comparative examples 1 and 2, the measurement results are shown in Table I and Table II, and the extraction yields of DNA and RNA can be calculated from the measured concentrations and known volumes, respectively.
The paraffin-embedded tissue DNA NanoDrop test results are shown in Table I.
List one
The results of RNA NanoDrop detection of paraffin-embedded tissues are shown in Table II.
Watch II
As can be seen from tables I and II, the DNA and RNA yields obtained by dewaxing using dewaxing system 10 were significantly higher than those obtained by dewaxing with xylene, and furthermore, the operation time was shortened by about 50% compared with that obtained by dewaxing with xylene using dewaxing system 10.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way; those skilled in the art can smoothly practice the invention as shown in the drawings and described above; however, those skilled in the art will appreciate that many modifications, adaptations, and variations of the present invention are possible in light of the above teachings without departing from the scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the present invention.
Claims (8)
1. The high-intensity focused ultrasound dewaxing system for paraffin embedded tissues is characterized by comprising a single-chip machine (12), a signal source circuit (13), a power supply (14), a power amplifying circuit (15) and a focusing piezoelectric transducer (17); the output end of the singlechip (12) is respectively connected with the signal source circuit (13) and the input end of the power supply (14); the output ends of the signal source circuit (13) and the power supply (14) are respectively connected with the input end of the power amplifying circuit (15); said power amplifying circuit (15)
The output end is connected with the input end of the focusing piezoelectric transducer (17); wherein,
the singlechip (12) controls the signal source circuit (13) to output two paths of square wave signals with opposite levels and frequency equal to the resonance frequency of the focusing piezoelectric transducer (17), and the square wave signals pass through the singlechip
(12) After PWM modulation, the PWM signals are respectively output from an OUTA end and an OUTB end of the signal source circuit (13);
the power amplifying circuit (15) comprises a first high-speed optocoupler (151), a second high-speed optocoupler (152), a grid driver (153), a current-limiting resistor R1, a current-limiting resistor R2, an NMOS tube Q1, an NMOS tube Q2,
A radio frequency choke RFC, a high frequency transformer T1;
the OUTA end and the OUTB end of the signal source circuit (13) are respectively connected with the first high-speed optocoupler (151) and the second high-speed optocoupler (152); the output end of the first high-speed optocoupler (151) and the output end of the second high-speed optocoupler (152) are respectively connected with the two input ends of the grid driver (153); the OUTA end of the grid driver (153) is sequentially connected with the current limiting resistor R1 and the NMOS tube Q1; the OUTB end of the gate driver (153) is sequentially connected with the current limiting resistor R2 and the transistor
NMOS tube Q2;
the power supply (14) is a programmable digital voltage source which in turn passes through the radio frequency choke RFC,
The high-frequency transformer T1 is connected to a circuit where the NMOS tube Q1 and the NMOS tube Q2 are positioned respectively;
the power amplifying circuit (15) is used for voltage gating and amplifying the signals output by the OUTA end and the OUTB end, outputting the signals to the focusing piezoelectric transducer (17), and driving the focusing piezoelectric transducer (17) to generate PWM modulated ultrasonic excitation so as to enable a sample positioned in a focusing area
The liquid in the tube is vibrated, so that paraffin in the sample tube is crushed and emulsified.
2. The high-intensity focused ultrasound dewaxing system for paraffin embedded tissues according to claim 1, further comprising an upper computer (11) electrically connected with the single chip microcomputer (12),
is used for receiving operation instructions and sending the instructions to the singlechip (12).
3. The high intensity focused ultrasound dewaxing system for paraffin embedded tissue according to claim 1, further comprising an impedance matching structure (16) connected to the power amplifying circuit (15) output; the impedance matching structure (16) is connected with the focusing piezoelectric transducer (17)
And the impedance of the focusing piezoelectric transducer (17) is matched.
4. A high intensity focused super-imaging device for paraffin-embedded tissue as claimed in any one of claims 1-3
Acoustic dewaxing system, characterized in that the signal source circuit (13) comprises:
a direct digital frequency synthesis module (131) for outputting signals of opposite levels and equal frequency to the focus
Two square wave signals of resonant frequency of the piezoelectric transducer (17);
and the two digital AND gates are used for performing AND operation on the square wave signal and the PWM wave signal.
5. A high intensity focused ultrasound dewaxing system for paraffin embedded tissue according to any of claims 1 to 3 wherein the resonance frequency of said focused piezoelectric transducer (17) is 100 kHz-100 kHz
10MHz。
6. Dewaxing apparatus comprising a use according to any one of claims 1 to 5
A high intensity focused ultrasound dewaxing system for paraffin embedded tissue.
7. A dewaxing device according to claim 6 comprising a container (21) provided with a cavity (211), a holder (212); the cavity (211) is filled with ultrasonic transmission
A medium; the fixing frame (212) is used for fixing the sample tube (30);
the focusing piezoelectric transducer (17) is fixed in the accommodating cavity (211); said sample tube (30)
Is located in a focal region of the focusing piezoelectric transducer (17).
8. The dewaxing apparatus as recited in claim 7 wherein said focusing piezoelectric element
The transducer (17) is fixed at the bottom of the cavity (211).
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| CN2153176Y (en) * | 1993-05-14 | 1994-01-12 | 曹鹏远 | Ultrasonic power supply |
| US5703443A (en) * | 1996-03-29 | 1997-12-30 | Samsung Electronics Co., Ltd. | Horizontal deflection output circuit |
| US6719449B1 (en) * | 1998-10-28 | 2004-04-13 | Covaris, Inc. | Apparatus and method for controlling sonic treatment |
| CN216978545U (en) * | 2021-10-19 | 2022-07-15 | 中国科学院苏州生物医学工程技术研究所 | High-intensity focused ultrasound dewaxing system and dewaxing device for paraffin-embedded tissues |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9080167B2 (en) * | 2012-11-16 | 2015-07-14 | Covaris, Inc. | System and method for processing paraffin embedded samples |
| US9335237B2 (en) * | 2013-06-06 | 2016-05-10 | Covaris, Inc. | Systems and methods for acoustically processing tissues samples |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2153176Y (en) * | 1993-05-14 | 1994-01-12 | 曹鹏远 | Ultrasonic power supply |
| US5703443A (en) * | 1996-03-29 | 1997-12-30 | Samsung Electronics Co., Ltd. | Horizontal deflection output circuit |
| US6719449B1 (en) * | 1998-10-28 | 2004-04-13 | Covaris, Inc. | Apparatus and method for controlling sonic treatment |
| CN216978545U (en) * | 2021-10-19 | 2022-07-15 | 中国科学院苏州生物医学工程技术研究所 | High-intensity focused ultrasound dewaxing system and dewaxing device for paraffin-embedded tissues |
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