Remote biological tissue transparentizing system and method

Abstract

The invention relates to the technical field of biological tissue transparentizing, in particular to a remote biological tissue transparentizing system and method. The remote biological tissue transparentizing system comprises: a sample cell for accommodating biological tissue; the liquid storage bottle is used for containing a reagent; a pump for pumping the reagent; a first conduit for connecting the sample cell and the pump; the second guide pipe is used for connecting the pump and the liquid storage bottle, and an electromagnetic valve and a flow meter are arranged on the second guide pipe; the PLC is electrically connected with the electromagnetic valve, the flow meter and the pump, and the PLC is used for obtaining flow data from the flow meter and controlling the electromagnetic valve and the pump; and the remote operation system is connected with the PLC and is used for remotely controlling the PLC. By means of the remote biological tissue transparentizing system, biological tissue transparentizing can be achieved remotely.

Description

Technical Field

[0001] The present invention relates to the technical field of biological tissue transparency, and in particular to a remote biological tissue transparency system and method. Background Art

[0002] Tissue clearing has been widely used in biological research, especially in neurobiological research, which allows researchers to obtain overall morphological data with the help of light sheet microscopy without traditional sectioning process.

[0003] Commonly used tissue clearing techniques include water-based (such as SeeDB, FRUIT, Scale, CUBIC), organic solvent-based (such as Disco series, BABB, PEGASOS), hydrogel-based (such as CLARITY, SHIELD, PACT) and other clearing methods. These methods have their own characteristics in specific operations: the reagent formula of the water-based clearing method is simple, and the clearing effect is average; the hydrogel-based clearing method has a good clearing effect, but requires special instruments and equipment; the organic solvent-based clearing method has simple reagents and good clearing effects, but the reagents themselves have certain toxicity (Table 1). However, in actual operation, most of these methods rely on manual operation, which requires high experimental experience of the operator, and is very cumbersome and time-consuming.

[0004] Table 1 Comparison of various transparency methods There are already some automated transparent devices, but these devices lack remote control functions, and the waste liquid discharged has the disadvantage of evaporating into the air and affecting the health of the experimenters. Summary of the invention

[0005] Based on this, the present invention provides a remote biological tissue clearing system and method, which at least solves one problem in the prior art.

[0006] In a first aspect, the present invention provides a remote biological tissue clearing system, comprising: A sample cell for containing biological tissue; Reservoir bottles for containing reagents; a pump for pumping reagents; a first conduit for connecting the sample cell and the pump; a second conduit for connecting the pump and the liquid storage bottle, wherein the second conduit is provided with a solenoid valve and a flow meter; A PLC controller electrically connected to the solenoid valve, the flow meter and the pump, respectively, the PLC controller being used to obtain flow data from the flow meter and control the solenoid valve and the pump; and A remote operating system connected to a PLC controller, the remote operating system is used to remotely control the PLC controller.

[0007] In a second aspect, the present invention provides a remote biological tissue clearing method, which uses the above-mentioned remote biological tissue clearing system and comprises the following steps: Putting biological tissue into the sample pool; Add reagent into the storage bottle; The PLC controller is controlled by a remote operating system, thereby operating the solenoid valve and the pump through the PLC controller, so that the reagent flows from the liquid storage bottle into the sample pool or flows out from the sample pool to the liquid storage bottle.

[0008] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects: (1) The biological tissue transparency experiment can be completed remotely and intelligently. The operator only needs to put the biological tissue sample into the sample pool and set the relevant experimental process. After the program is completed, a completely transparent sample can be obtained, which can greatly reduce the operator's stay time at the experimental site; (2) The reagents were improved by using gradient ethanol-water solutions for dehydration to reduce the toxic effects of methanol volatilization; and a waste gas removal scheme was adopted to reduce the impact of organic solvents on the health of operators; (3) A transparent solution that is compatible with both water-based and organic solvent-based solutions; (4) Dozens of samples can be placed in the sample pool at one time, significantly improving the efficiency of biological tissue transparency experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the overall structure of the remote biological tissue transparentization system in an embodiment of the present invention.

[0010] Figure 2 Schematic diagram of the experimental process of Example 1 of the present invention.

[0011] Figure 3 This is a normal bright field photograph of the mouse brain sample before and after transparentization in Example 1 of the present invention, wherein the left side is before transparentization and the back side is after transparentization.

[0012] Figure 4 This is a vascular immunofluorescence photograph of the mouse brain sample after transparency in Example 1 of the present invention.

[0013] Figure 5 This is a fluorescence photograph of the eye blood vessels of the mouse brain sample after transparency in Example 1 of the present invention.

[0014] Figure 6 This is a neuronal fluorescence image of the mouse brain sample after transparency in Example 3 of the present invention. DETAILED DESCRIPTION

[0015] The technical solution of the present invention will be described in detail below in conjunction with the specific implementation methods of the present invention. However, the following embodiments are only used to understand the present invention and cannot limit the present invention. The embodiments and features in the embodiments of the present invention can be combined with each other. The present invention can be implemented in a variety of different ways as defined and covered by the claims.

[0016] In a first aspect, the present invention provides a remote biological tissue transparentization system. Figure 1 The schematic diagram of its structure includes: A sample pool 116 for containing biological tissue; A liquid storage bottle 111 for containing reagents; a pump 114 for pumping reagents; A first conduit 115 for connecting the sample cell 116 and the pump 114; A second conduit 122 for connecting the pump 114 and the liquid storage bottle 111, wherein the second conduit 122 is provided with a solenoid valve 112 and a flow meter 113; A PLC controller electrically connected to the solenoid valve 112, the flow meter 113 and the pump 114, respectively, the PLC controller being used to obtain flow data from the flow meter 113 and control the solenoid valve 112 and the pump 114; and A remote operating system connected to a PLC controller, the remote operating system is used to remotely control the PLC controller.

[0017] Since the sample pool, pump and liquid storage bottle are connected in sequence through the first conduit and the second conduit, and the second conduit is provided with a solenoid valve and a flow meter, the flow data can be obtained from the flow meter through the PLC controller and the action of the solenoid valve and the pump can be controlled. After reaching the set flow, the feedback signal triggers the PLC controller to close the corresponding solenoid valve, thereby automatically controlling the reagent to flow into or out of the sample pool, and realizing the automated biological tissue transparency operation. Moreover, by setting up a remote operating system, the PLC controller can be remotely controlled, thereby realizing the remote control of the biological tissue transparency operation.

[0018] In some embodiments, a quick connector is provided at the top of the sample pool 116. For example, the sample pool 116 can be a 50 mL centrifuge tube or a reagent bottle for ordinary experiments; it can be a light-transmitting reagent bottle or a light-proof reagent bottle. The quick connector is used to conveniently connect the catheter. Optionally, the first catheter 115 extends into the bottom of the sample pool, and the first catheter 115 is cut off at the position where the first catheter 115 is located at the top of the sample pool 116, and a quick connector is installed at the cut position to facilitate the connection between the catheter inside the sample pool 116 and the catheter outside the sample pool 116, thereby ensuring that the liquid in the sample pool 116 is clean.

[0019] In some embodiments, a rotating rod 118 is provided in the sample pool 116, and a sample box 117 is suspended at the bottom of the rotating rod 118. An electric motor is provided above the rotating rod 118, and the electric motor is electrically connected to the PLC controller; the PLC controller drives the rotating rod 118 to rotate through the electric motor to drive the sample box 117 to rotate, so as to achieve the purpose of shaking and cleaning the biological tissue in the sample box 117. The sample box 117 is a metal box or a plastic box for temporarily storing biological tissue samples. There are holes around the sample box 117 to facilitate the immersion of reagents into the biological tissue samples in the sample box 117. Optionally, the number of sample boxes 117 can be multiple to improve experimental efficiency.

[0020] In some embodiments, the remote biological tissue clearing system further includes a temperature control station 119 electrically connected to the PLC controller, and the temperature control station 119 is used to control the temperature in the sample pool 116 .

[0021] In some embodiments, a quick connector is provided at the top of the liquid storage bottle 111. For example, the liquid storage bottle 111 may be a 50 ml centrifuge tube or a reagent bottle for a common experiment. Optionally, the second conduit 122 extends into the bottom of the liquid storage bottle 111, and the second conduit 122 is cut off at the position where the second conduit 122 is located at the top of the liquid storage bottle 111, and a quick connector is installed at the cut position to facilitate the connection between the inner conduit of the liquid storage bottle 111 and the outer conduit of the liquid storage bottle 111.

[0022] In some embodiments, the number of liquid storage bottles 111 can be multiple, so as to store different reagents. For example, the number of liquid storage bottles 111 is ten, nine of which are used to store reagents and one is used to store waste liquid. The number of second conduits 122 is equal to the number of liquid storage bottles 111, and each liquid storage bottle 111 is connected to the pump 114 through the second conduit 122, and each second conduit 122 is provided with a solenoid valve 112 and a flow meter 113, and all solenoid valves 112 and flow meters 113 are connected to the PLC controller. The pump 114 has the function of forward rotation and reverse rotation. When the pump 114 rotates forward, the reagent in the liquid storage bottle 111 flows into the sample pool 116 through the second conduit 122 and the first conduit 115; when the pump 114 reverses, the reagent in the sample pool 116 is recovered to the corresponding liquid storage bottle 111 through the first conduit 115 and the second conduit 122, and the path of reagent recovery is controlled by the solenoid valve 112.

[0023] In some embodiments, the remote biological tissue clearing system further includes an exhaust gas adsorption device 121 and an exhaust fan 120. The exhaust fan 120 is used to remove harmful substances volatilized from the sample pool 116. The exhaust fan 120 is connected to the exhaust gas adsorption device 121 to reduce the exhaust gas emission into the environment.

[0024] In some embodiments, the remote operating system includes an IoT signal generator 201 and a mobile terminal 202, wherein the IoT signal generator 201 is connected to a PLC controller, and the mobile terminal 202 is communicatively connected to the IoT signal generator 201. Optionally, the IoT signal generator 201 is communicatively connected to the mobile terminal 202 via wifi, 4G, 5G or Ethernet; the mobile terminal 202 is used to view the operating status of the remote biological tissue transparency system in real time, and can directly control each specific parameter.

[0025] In some embodiments, the PLC controller includes a touch screen 101 and a circuit controller 102. The circuit controller 102 is connected to components such as a pump 114, a solenoid valve 112, a flow meter 113, a rotating rod 118, a temperature control station 119, an IoT signal generator 201, and an exhaust fan 120 to control these components. The touch screen 101 is an operation interface, and buttons can be pressed. The operation interface in the touch screen 101 and the remote operation interface on the mobile terminal 202 can have the same function and appearance. The mobile terminal 202 can be a mobile phone. During the operation of the system, the relevant process can be viewed and monitored in real time through the mobile phone.

[0026] In some embodiments, the remote biological tissue clearing system further includes a frame 103, and the frame 103 is used to accommodate other components except the mobile terminal 202. Optionally, the frame 103 includes an upper layer, a middle layer and a lower layer, the bottom layer of the frame 103 is used to place a liquid storage bottle 111, the middle layer of the frame 103 is used to place a pump 114, a solenoid valve 112, and a flow meter 113, and the upper layer of the frame 103 is used to place a PLC controller, a sample pool 116, an exhaust gas adsorption device 121 and an exhaust fan 120; the touch screen 101 of the PLC controller is embedded in the frame 103.

[0027] In a second aspect, the present invention provides a remote biological tissue clearing method, which uses a remote biological tissue clearing system, comprising the following steps: Putting biological tissue into the sample pool 116; Putting reagent into the liquid storage bottle 111; The PLC controller is controlled by a remote operating system, thereby operating the solenoid valve 112 and the pump 114 through the PLC controller, so that the reagent flows from the liquid storage bottle 111 into the sample pool 116 or flows out from the sample pool 116 into the liquid storage bottle 111 .

[0028] In some embodiments, the reagents include PBS, 20% alcohol, 40% alcohol, 60% alcohol, 80% alcohol, 100% alcohol, a mixture of DCM and 100% alcohol, DCM, and DBE (diBenzyl Ether) solution, and are respectively placed in storage bottles. 20% alcohol refers to alcohol with a volume ratio of 20%, 40% alcohol refers to alcohol with a volume ratio of 40%, and so on. The volume ratio of DCM to 100% alcohol in the mixture of DCM and 100% alcohol can be 2:1.

[0029] In some embodiments, the PLC controller operates the solenoid valves and pumps through the following program: PBS flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 20% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 40% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 60% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 80% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 100% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 100% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; a mixture of DCM and 100% alcohol flows in for 2 minutes, waits for 12 hours, and flows out for 2 minutes; DCM flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; DCM flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; DBE solution flows in for 2 minutes and stops.

[0030] Some typical embodiments are described below.

[0031] Example 1 The whole mouse brain was remotely transparentized using the iDISCO+ method. The steps are as follows: (1) Mouse brain sampling: Anesthetize the mouse, perfuse the left ventricle with 20ml PBS, and fix the left ventricle with 20ml 4% PFA; dissect the brain, remove the whole brain, and fix it with 4% PFA at 4℃ overnight; the next day, wash it with PBS at room temperature for 30min*3 times with shaking; (2) Using the remote biological tissue clearing system to clear the mouse brain: Place the required reagents in the storage bottle and put the mouse brain in the sample box; dehydrate with 0%, 20%, 40%, 60%, 80%, 100%, and 100% MeOH gradient, 1 hour / time, and shake at room temperature; incubate with a mixture of DCM and MeOH with a volume ratio of 2:1 at room temperature for 18 hours; wash with 100% DCM on a shaker for 1 hour*2 times to wash away the methanol; incubate with DBE, let it stand until the tissue is transparent, and store it in DBE at room temperature; (3) Take out the transparent mouse brain sample and take a microscopic photo.

[0032] The experimental process of this embodiment is shown in FIG. Figure 2 , you only need to manually put in the biological tissue sample, and it will be automatically transparent. After transparentization, the biological tissue sample is manually taken out, and the manual participation time is about 10 minutes. The ordinary bright field photos of the mouse brain sample before and after transparentization in this embodiment are as follows Figure 3 As shown, the immunofluorescence photos of mouse brain blood vessels are shown in Figure 4 As shown, the fluorescent photos of the blood vessels in the mouse eye are as follows Figure 5 shown.

[0033] Example 2 The mouse hemibrain was remotely cleared and processed for immunohistochemistry using the iDISCO+ method. The steps are as follows: (1) Mouse brain sampling: Anesthetize the mouse, perfuse the left ventricle with 20ml PBS, and fix the left ventricle with 20ml 4% PFA; dissect the brain, remove the whole brain, and divide the left and right hemispheres; fix with 4% PFA at 4℃ overnight; the next day, wash with PBS at room temperature for 30min*3 times with shaking; (2) Pretreatment of mouse brain using remote biological tissue clearing system: Place the required reagents in the storage bottle and place the mouse brain in the sample box; dehydrate with 0%, 20%, 40%, 60%, 80%, 100%, 100% MeOH gradient, 1h / time; incubate overnight with a mixture of DCM and MeOH with a volume ratio of 2:1 at room temperature (RT); wash with 100% MeOH at room temperature for 1h*2 times, and cool at 4℃; incubate the sample with 5% H2O2 (diluted with MeOH) at 4℃ overnight; rehydrate with 80%, 60%, 40%, 20%, 0% MeOH gradient at room temperature for 1h / time; rinse with PBX (PBS / 3% Triton X100) at room temperature for 1h*2 times; (3) Immunohistochemistry of mouse brain using the remote biological tissue clearing system: Incubate samples in permeabilization solution for 36 hours at 37°C; block in blocking solution for 48 hours at 37°C; incubate with primary antibody (diluent: PBS / 3% Triton X100 / 8% donkey serum) for 14 days at 37°C; wash with PBX 4-5 times at 24 hours at RT; incubate with secondary antibody (diluent: PBS / 3% Triton X100 / 8% donkey serum) for 7 days at 37°C; wash with PBX 4-5 times at 24 hours at RT; (4) Use the remote biological tissue clearing system to clear the mouse brain after immunohistochemistry treatment: Dehydrate with 0%, 20%, 40%, 60%, 80%, 100%, 100% MeOH gradient, 1h / time; incubate with a mixture of DCM and MeOH with a volume ratio of 2:1 at room temperature overnight; wash with 100% DCM on a shaker for 1h*2 times to wash away the methanol; incubate with DBE and let stand until the tissue is transparent; (5) Take out the transparent mouse brain sample and take microscopic photos.

[0034] Example 3 The iDISCO+ modified method based on ethanol dehydration was used to remotely clear the whole mouse brain. The steps are as follows: (1) Mouse brain sampling: Anesthetize the mouse, perfuse the left ventricle with 20ml PBS, and fix the left ventricle with 20ml 4% PFA; dissect the brain, remove the whole brain, and fix it with 4% PFA at 4℃ overnight; the next day, wash it with PBS at room temperature for 30min*3 times with shaking; (2) Using the remote biological tissue clearing system to clear the mouse brain: Place the required reagents in the storage bottle and place the mouse brain in the sample box; dehydrate with 0%, 20%, 40%, 60%, 80%, 100%, 100% ethanol (EOH) gradient, 1h / time, RT; incubate with a mixture of DCM and EOH with a volume ratio of 2:1 at room temperature overnight; wash with 100% DCM on a shaker for 15min*2 times to wash off the ethanol; incubate with DBE and let stand until the tissue is transparent; (3) Take out the transparent mouse brain sample and take a microscopic photo.

[0035] The neuronal fluorescence image of the mouse brain sample after transparency in this example is as follows Figure 6 shown.

[0036] Example 4 The whole mouse brain was remotely transparentized based on the PEGASOS method. The steps are as follows: (1) Mouse brain sampling: Anesthetize the mouse, perfuse the left ventricle with 20ml PBS, and fix the left ventricle with 20ml 4% PFA; dissect the brain, remove the whole brain, and fix it with 4% PFA at 4℃ overnight; the next day, wash it with PBS at room temperature for 30min*3 times with shaking; (2) Using the remote biological tissue clearing system to clear the mouse brain: The required reagents were placed in the storage bottle, and the mouse brain was placed in the sample box; the samples were washed with 25% Quadrol (#122262, Sigma-Aldrich) at 37°C for 2 days with shaking, and the solution was changed once a day; the mouse brain samples with darker colors were treated with 3% ammonia solution for 6 hours; the samples were washed with 30% tert-butyl alcohol + 3% Quadrol, 50% tert-butyl alcohol + 3% Quadrol, 70% tert-butyl alcohol + 3% Quadrol, and 70% tert-butyl alcohol + 30% PEG-MMA-500 (#447943, Sigma-Aldrich) + 3% Quadrol for 1 day with shaking; 70% v / v tert-butyl alcohol, 27% v / v PEG-MMA-500 and 3% w / v Dehydrate with tB-PEG solution composed of Quadrol for 2 days, changing the solution once a day; immerse the dehydrated tissue samples in BB-PEG (#B6630, Sigma-Aldrich) clearing medium at 37°C for at least 1 day until the tissue is completely transparent; (3) Take out the transparent mouse brain sample and take a microscopic photo.

[0037] Example 5 The whole mouse brain was remotely transparentized based on the ScaleCUBIC method. The steps are as follows: (1) Mouse brain sampling: Anesthetize the mouse, perfuse the left ventricle with 20ml PBS, and fix the left ventricle with 20ml 4% PFA; dissect the brain, remove the whole brain, and fix it with 4% PFA at 4℃ overnight; the next day, wash it with PBS at room temperature for 30min*3 times with shaking; (2) Using the remote biological tissue clearing system to clear the mouse brain: Place the required reagents in the storage bottle and place the mouse brain in the sample box; wash with ScaleCUBIC-1 (25 wt% urea + 25 wt% tetrahydroxypropylethylenediamine (EDTP) + 15 wt% Triton X-100) by shaking for 3 days, changing the solution every day; immerse in fresh ScaleCUBIC-1 for another 3-4 days; wash with 20 wt% sucrose (diluted with PBS) by shaking for 3 times, 30 minutes each time; wash with ScaleCUBIC-2 (50 wt% sucrose + 25 wt% urea + 10 wt% triethanolamine + 0.1% Triton X-100) by shaking for 3-7 days, changing the solution every 2 days; wash with 20% sucrose (PBS) by shaking for 3 times, 30 minutes each time; store in OCT (#4583, Sakura) until the tissue is completely transparent; (3) Take out the transparent mouse brain sample and take a microscopic photo.

[0038] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same or equivalent means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. A remote biological tissue clearing system, characterized in that: include: A sample cell for containing biological tissue; Reservoir bottles for containing reagents; a pump for pumping reagents; a first conduit for connecting the sample cell and the pump; a second conduit for connecting the pump and the liquid storage bottle, wherein the second conduit is provided with a solenoid valve and a flow meter; A PLC controller electrically connected to the solenoid valve, the flow meter and the pump, respectively, the PLC controller being used to obtain flow data from the flow meter and control the solenoid valve and the pump; and A remote operating system connected to a PLC controller, the remote operating system is used to remotely control the PLC controller.

2. The remote biological tissue clearing system according to claim 1, characterized in that: A rotating rod is arranged in the sample pool, and a sample box is hung at the bottom end of the rotating rod.

3. The remote biological tissue clearing system according to claim 1, characterized in that: The remote biological tissue clearing system also includes a temperature control station electrically connected to the PLC controller, and the temperature control station is used to control the temperature in the sample pool.

4. The remote biological tissue clearing system according to claim 1, characterized in that: There are multiple storage bottles to store different reagents.

5. The remote biological tissue clearing system according to claim 1, characterized in that: The remote biological tissue clearing system also includes an exhaust gas adsorption device and an exhaust fan.

6. The remote biological tissue clearing system according to claim 1, characterized in that: The remote operating system includes an Internet of Things signal generator and a mobile terminal. The Internet of Things signal generator is electrically connected to the PLC controller, and the mobile terminal is communicatively connected to the Internet of Things signal generator.

7. The remote biological tissue clearing system according to claim 1, characterized in that: The PLC controller includes a touch display screen and a circuit controller.

8. A remote biological tissue clearing method, using the remote biological tissue clearing system according to any one of claims 1 to 7, comprising the following steps: Putting biological tissue into the sample pool; Add reagent into the storage bottle; The PLC controller is controlled by a remote operating system, thereby operating the solenoid valve and the pump through the PLC controller, so that the reagent flows from the liquid storage bottle into the sample pool or flows out from the sample pool to the liquid storage bottle.

9. The method according to claim 8, characterized in that Reagents include PBS, 20% alcohol, 40% alcohol, 60% alcohol, 80% alcohol, 100% alcohol, a mixture of DCM and 100% alcohol, DCM, and DBE solution, and are respectively packed into storage bottles.

10. The method according to claim 9, characterized in that The PLC controller operates the solenoid valves and pumps through the following program: PBS flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 20% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 40% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 60% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 80% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 100% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; 100% alcohol flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; a mixture of DCM and 100% alcohol flows in for 2 minutes, waits for 12 hours, and flows out for 2 minutes; DCM flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; DCM flows in for 2 minutes, waits for 1 hour, and flows out for 2 minutes; DBE solution flows in for 2 minutes and stops.

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