Drosophila melanogaster automatic feeding coupling two-photon imaging system

Abstract

The invention discloses a coupled two-photon imaging system for automatic feeding of drosophila melanogaster. The system mainly comprises four components, namely a CL-01A stepping motor controller, a three-axis stepping motor, a micro-injection pump controller and an acrylic injector propeller. The operation process is as follows: the CL-01A controls the stepping motor to move, and the needle head is moved to a proper position right in front of the drosophila melanogaster; the micro-injection pump controller controls the acrylic injector to inject a real object; the stepping motor stays for a period of time, and continuous feeding stimulation is given; the CL-01A controls the stepping motor to return to the original position, and fruit fly feeding is finished. The system realizes the automatic control of the behavior of the drosophila melanogaster to take food, and solves the problems of taking food and recording the behavior of the drosophila melanogaster while recording the distribution of neurons in a narrow space under a two-photon microscope.

Description

Technical Field

[0001] The invention relates to the technical field of fruit fly experimental devices, and in particular to a fruit fly automatic feeding coupled two-photon imaging system. Background Art

[0002] Drosophila is a classic model organism with advantages such as short breeding cycle and high reproduction rate. In addition, Drosophila has highly conserved genes, behavioral logic and metabolic pathways similar to those of mammals, a relatively simple nervous system and abundant genetic tools, making it an important model for studying animal instinctive behavior.

[0003] In vivo two-photon calcium imaging is a technique used to record neuronal activity in the intact brain. It works on the basis that when a neuron fires an action potential, intracellular calcium levels rise, which can be detected using fluorescent molecules that bind to calcium, thus allowing neuronal activity to be recorded.

[0004] To study the coupling relationship between Drosophila neurons and behaviors, it is necessary to use a two-photon microscope and a camera to record Drosophila neuronal activity and behavioral changes respectively under a specific genetic background or artificial treatment. This theory will encounter the following problems in specific practice:

[0005] (1) The space under the two-photon microscope is relatively small, so a system suitable for the two-photon microscope needs to be built

[0006] (2) How to align the recorded neuronal and behavioral changes in time scale?

[0007] (3) Giving fruit flies feeding stimulation is a very delicate operation in three-dimensional space. How to control the horizontal distance and time of feeding / socializing?

[0008] (4) Manually completing the operation of moving forward, stopping, and withdrawing may lack rigor. How to introduce an automatic control system to standardize the entire process?

[0009] (5) When feeding, how to control the size of food droplets so that fruit flies can eat by extending their proboscis normally, while also avoiding sticky and overly large droplets that hinder the normal movement of fruit flies? Summary of the invention

[0010] Aiming at the shortcomings of in vitro experimental technology for fruit flies in an active state and the difficulty in simultaneously recording neuronal and behavioral activities, the present invention provides a fruit fly automatic feeding coupled two-photon imaging system.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] A fruit fly automatic feeding coupled two-photon imaging system, comprising a microinjection pump controller, a stepper motor controller, a syringe propulsion device, a syringe, a stepper motor, a feeding needle, a two-photon microscope and a camera;

[0013] The microinjection pump controller is connected to the syringe propulsion device, the microinjection pump controller provides propulsion power for the syringe propulsion device, and can adjust the propulsion direction and propulsion power of the syringe propulsion device; the syringe is arranged on the syringe propulsion device, and is filled with nutrient solution, and the syringe is connected to the input end of the feeding needle through a hose; the feeding needle is arranged on the stepping motor through a slider, and the output end of the feeding needle is facing the mouthparts of the experimental fruit fly;

[0014] The stepper motor is connected to the stepper motor controller via a wire; the stepper motor controller is pre-set with driving parameters, which can adjust the driving direction and driving force of the stepper motor;

[0015] The lenses of the two-photon microscope and the camera are both aimed at the experimental fruit flies; the camera is used to record the behavior of the fruit flies, and the two-photon microscope is used to record the fluorescence changes of the calcium signals of the neurons in the fruit fly brain when the fruit flies are active.

[0016] As a preferred solution of the present invention, the feeding needle is connected to the slider via a knob, and the feeding needle can rotate with the knob.

[0017] The present invention also provides an imaging method using the above-mentioned fruit fly automatic feeding coupled two-photon imaging system, comprising the following steps:

[0018] 1) placing a syringe filled with nutrient solution on a syringe propulsion device and connecting it to an input end of a feeding needle through a hose;

[0019] 2) Open a window on the head of the experimental fruit fly and fix it, then place the experimental fruit fly under the lens of a two-photon microscope and camera;

[0020] 3) Adjust the angle of the feeding needle so that the output end of the feeding needle is facing the head of the experimental fruit fly;

[0021] 4) Observe the feeding needle set on the stepper motor through the camera, debug the stepper motor controller, and determine the movement end point of the feeding needle, and then let the stepper motor retreat back to the starting point;

[0022] 5) Debug the microinjection pump controller so that each time the syringe propulsion device propels the syringe, the nutrient solution squeezed out of the syringe into the feeding needle will not hinder the movement of the experimental fruit flies;

[0023] 6) Turn on the camera and two-photon microscope to record the behavior of the experimental fruit flies and the changes in the calcium signals of the experimental fruit flies' neurons;

[0024] 7) Control the stepper motor controller and the microinjection pump controller to feed, the experimental fruit flies start to eat, and the camera and two-photon microscope record the experimental fruit flies eating and the changes in the calcium signals of the experimental fruit flies' neurons;

[0025] 8) When the set time is up, the stepper motor controller drives the stepper motor to return to its original position, the fruit fly finishes eating, the camera and the two-photon microscope stop recording, and imaging is completed.

[0026] As a preferred embodiment of the present invention, in the step 2), a window is opened on the head of the test fruit fly, the head of the test fruit fly is fixed, and the fruit fly is placed on a floating ball that can rotate freely.

[0027] As a preferred embodiment of the present invention, determining the movement end point of the feeding needle in step 4) is specifically as follows: debugging the stepper motor controller and observing the feeding needle disposed on the stepper motor through a macro camera so that the movement end point of the feeding needle meets the feeding distance relative to the position of the experimental fruit fly with its head fixed.

[0028] As a preferred embodiment of the present invention, the step 7) of controlling the stepper motor controller and the microinjection pump controller to perform feeding is specifically as follows: starting the stepper motor controller to drive the slider on the stepper motor to move toward the experimental fruit fly so that the feeding needle reaches the end of the movement; then starting the microinjection pump controller to control the syringe propulsion device to push the syringe, so that the nutrient solution enters the feeding needle from the syringe and seeps out from the output end of the feeding needle.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention uses two controllers and two actuators to realize the automatic forward and backward movement of the mechanical arm, the stop and the injection of the syringe. The implementation method is simple and flexible, and the parameters can be adjusted to adapt to different fruit flies and different experimental schemes, ultimately achieving the purpose of automatic feeding. In addition, the device is used in combination with two-photon and posture analysis methods to realize the study of the neuronal firing and specific behavior patterns of fruit flies. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the automatic feeding system in this device;

[0032] Figure 2 It is the feeding process of this device on the crawling ball under two photons;

[0033] Figure 3 It is the result of simultaneous two-photon recording of neuronal activity and fruit fly behavior pattern processed by posture analysis software;

[0034] Figure 4This is an overview of the device using a camera to record the experiment under two-photon conditions. DETAILED DESCRIPTION

[0035] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0036] The present invention will be described in detail below with reference to the accompanying drawings.

[0037] like Figure 1 and Figure 4 As shown, the fruit fly automatic feeding coupled two-photon imaging system provided by the present invention includes a microinjection pump controller 3, a stepper motor controller 1, a syringe propulsion device 4, a syringe 6, a stepper motor 2, a feeding needle 9, a two-photon microscope and a camera; the microinjection pump controller 3 is connected to the syringe propulsion device 4, the microinjection pump controller 3 provides propulsion power for the syringe propulsion device 4, and can adjust the propulsion direction and propulsion power of the syringe propulsion device 4; the syringe 6 is arranged on the syringe propulsion device 4, and is filled with nutrient solution. The syringe is connected to the input end of the feeding needle through a hose, and the tail of the syringe is fixedly connected to the propulsion plate 11 of the syringe propulsion device 4. The feeding needle 9 is arranged on the stepping motor through the slider 10, and the output end of the feeding needle is facing the mouthparts of the experimental fruit fly; in a specific embodiment of the present invention, the feeding needle is connected to the slider through two knobs, and the two knobs are respectively a horizontal knob 7 and a vertical knob 8. The feeding needle is aligned with the fruit fly in a horizontal position by rotating the horizontal knob 7, and the feeding needle is aligned with the fruit fly in a vertical position by rotating the vertical knob 8.

[0038] like Figure 2 As shown, an embodiment of applying the device to study the fear-seeking behavior pattern of fruit flies is as follows;

[0039] The stepper motor 2 is connected to the stepper motor controller 1 through a wire; the stepper motor controller 1 is pre-set with driving parameters, which can adjust the driving direction and driving force of the stepper motor 2; in this embodiment, the CL-01A stepper motor controller and the three-axis stepper motor are used;

[0040] The lenses of the two-photon microscope and the camera are both aimed at the experimental fruit flies; the camera is used to record the behavior of the fruit flies, and the two-photon microscope is used to record the fluorescence changes of the calcium signals of the neurons in the fruit fly brain when the fruit flies are active.

[0041] like Figure 1As shown, they are CL-01A stepper motor controller, three-axis stepper motor, micro-injection pump controller, and acrylic syringe pusher. Connect the wires correctly and turn on the power 1, and fill the syringe with food in advance;

[0042] A window was opened on the head of the experimental fruit fly, and then the head of the fruit fly was fixed under a two-photon microscope. The height of the climbing ball was adjusted so that the fruit fly could walk normally on the climbing ball.

[0043] The feeding needle is adjusted manually by turning the knob on the three-axis stepper motor so that the feeding needle is facing the fixed experimental fruit fly;

[0044] Before the formal experiment begins, the needle attached to the stepper motor is observed through a macro camera, and the end point of the movement is adjusted so that the position of the needle relative to the fixed fruit fly meets the experimental design. After the end point is determined, the stepper motor is moved back to the starting point. The debugging and return processes are achieved by moving the slider on the three-axis stepper motor left and right.

[0045] The formal experiment began, and the built-in program of the CL-01A stepper motor controller was edited; the microinjection pump controller selected the automatic mode and saved the pre-determined parameters, so that the two controllers could automatically drive the system to run in the next step; the camera and two-photon microscope were turned on to record the behavior of the fruit fly, the climbing ball, and the changes in the calcium signal of the fruit fly neurons;

[0046] The CL-01A controller drives the slider on the stepper motor to move toward the fruit fly, so that the feeding element on the stepper motor and the previously fixed fruit fly are in a suitable relative position; the CL-01A controller controls the stepper motor to stay at the end point for a predetermined time, so as to prepare for the fruit fly to continue feeding / socializing behavior;

[0047] The microinjection pump controller controls the acrylic syringe plate to advance, causing the syringe to start injecting, and a suitable volume of liquid penetrates into the space between the two small plates of the needle along the plastic hose; Figure 2 As shown, the fruit flies then start to eat until the set time is up, and the CL-01A controller controls the stepper motor to return to the starting position, and the fruit flies finish eating;

[0048] After the experiment is over, stop recording the two-photon and camera. Perform posture analysis and other processing on the recorded results to obtain the results of neuronal activity and fruit fly behavior patterns at the same time, such as Figure 3 As shown, the relationship between the behavior patterns of fruit flies and the firing of neurons can be analyzed.

[0049] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A fruit fly automatic feeding coupled two-photon imaging system, characterized in that: It includes a microinjection pump controller, a stepper motor controller, a syringe propulsion device, a syringe, a stepper motor, a feeding needle, a two-photon microscope, and a camera; The microinjection pump controller is connected to the syringe propulsion device; the microinjection pump controller provides propulsion power for the syringe propulsion device, and can adjust the propulsion power and the propulsion direction of the syringe propulsion device; the syringe is arranged on the syringe propulsion device, and is filled with nutrient solution, and the syringe is connected to the input end of the feeding needle through a hose; the feeding needle is arranged on the stepping motor through a slider, and the output end of the feeding needle is facing the mouthparts of the experimental fruit fly; The stepper motor is connected to the stepper motor controller via a wire; the stepper motor controller is pre-set with driving parameters, which can adjust the driving direction and driving force of the stepper motor; The two-photon microscope and the camera lens are both aimed at the experimental fruit fly; The camera is used to record the behavior of fruit flies, and the two-photon microscope is used to record the fluorescence changes of calcium signals in the neurons of the fruit fly brain when the fruit flies are active.

2. The fruit fly automatic feeding coupled two-photon imaging system according to claim 1, characterized in that: The feeding needle is connected to the slider via a knob, and the feeding needle can rotate along with the knob.

3. The fruit fly automatic feeding coupled two-photon imaging system according to claim 1, characterized in that: The syringe propulsion device is made of acrylic material.

4. An imaging method using the fruit fly automatic feeding coupled two-photon imaging system according to claim 1, characterized in that: The following steps are involved: 1) placing a syringe filled with nutrient solution on a syringe propulsion device and connecting it to an input end of a feeding needle through a hose; 2) Open a window on the head of the experimental fruit fly and fix it, then place the experimental fruit fly under the lens of a two-photon microscope and camera; 3) Adjust the angle of the feeding needle so that the output end of the feeding needle is facing the mouthparts of the experimental fruit fly; 4) Observe the feeding needle set on the stepper motor through the camera, debug the stepper motor controller, and determine the movement end point of the feeding needle, and then let the stepper motor retreat back to the starting point; 5) Debug the microinjection pump controller so that each time the syringe propulsion device propels the syringe, the nutrient solution squeezed out of the syringe into the feeding needle will not hinder the movement of the experimental fruit flies; 6) Turn on the camera and two-photon microscope to record the behavior of the experimental fruit flies and the changes in the calcium signals of the experimental fruit flies’ neurons; 7) Control the stepper motor controller and the microinjection pump controller to feed, the experimental fruit flies start to eat, and the camera and two-photon microscope record the experimental fruit flies eating and the changes in the calcium signals of the experimental fruit flies' neurons; 8) When the set time is up, the stepper motor controller drives the stepper motor to return to its original position, the fruit fly finishes eating, the camera and the two-photon microscope stop recording, and imaging is completed.

5. The imaging method of the fruit fly automatic feeding coupled two-photon imaging system according to claim 4, characterized in that: In the step 2), a window is opened on the head of the experimental fruit fly, the head of the experimental fruit fly is fixed, and the fruit fly is placed on a floating ball that can rotate freely.

6. The imaging method of the fruit fly automatic feeding coupled two-photon imaging system according to claim 4, characterized in that: Determining the movement end point of the feeding needle in step 4) is specifically as follows: debugging the stepper motor controller and observing the feeding needle disposed on the stepper motor through a camera macro, so that the movement end point of the feeding needle meets the feeding distance relative to the position of the experimental fruit fly with the head fixed.

7. The imaging method of the fruit fly automatic feeding coupled two-photon imaging system according to claim 4, characterized in that: In step 7), the step of controlling the stepper motor controller and the microinjection pump controller to perform feeding is specifically as follows: starting the stepper motor controller to drive the slider on the stepper motor to move toward the experimental fruit fly, so that the feeding needle reaches the end of the movement; then starting the microinjection pump controller to control the syringe propulsion device to push the syringe, so that the nutrient solution enters the feeding needle from the syringe and seeps out from the output end of the feeding needle.

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