Robotic platform for microinjection into single cells in brain tissue

Abstract Microinjection into single cells in brain tissue is a powerful technique to study and manipulate neural stem cells. However, such microinjection requires expertise and is a low‐throughput process. We developed the “Autoinjector”, a robot that utilizes images from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The Autoinjector enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. The Autoinjector successfully targets both apical progenitors (APs) and newborn neurons in the embryonic mouse and human fetal telencephalon. We used the Autoinjector to systematically study gap‐junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction‐coupled cluster. The throughput and versatility of the Autoinjector will render microinjection an accessible high‐performance single‐cell manipulation technique and will provide a powerful new platform for performing single‐cell analyses in tissue for bioengineering and biophysics applications.


I. Introduction
General microinjection guidelines are found in section VII and provide suggestions for parameter values. This user manual is designed to take you through the implementation of the automated microinjection system with the following assumptions: − Software is installed from github.com/ogshull/Autoinjector--You are using Sensapex manipulators, the custom pressure rig, and a Hamamatsu camera

II. Hardware Overview
The image above provides an overview of how the Autoinjector system operates with digital control shown in black and pneumatic (pressure) control shown in blue. All hardware components are controlled by the python guide user interface from the computer (GUI). In summary, the pressure is set by the user in the software which sends a signal to the Arduino (microcontroller) which in turn sets the pressure via the pressure control board and opens the valve (further discussion in section 5). The following page displays an indepth diagram and a schematic of the hardware.

III. Software Overview
The image above displays the guide user interface (GUI) written in python and can be launched from the desktop of the computer (see section V step 3 for further details). The parts are as follows: 1. Manipulator Calibrationthe calibration controls allow the user to calibrate the manipulator camera frames to those of the camera. Further instructions can be found in section V).

Draw
Trajectory -This is composed of the "Draw Edge" Button which allows the user to draw the desired trajectory (see section V steps 15 -18).
3. Display Settings -This allows you to control the gain of the microscope feed, and display or hide the trajectory you draw from the draw trajectory section 4. System Status -The system status updates the user with feedback from the interface and will report useful information such as parameter change updates, and errors.

Manipulator Controls -
The manipulator controls display the positions of the manipulator and allow you to advance the axes from the interface. Set the increment (in microns) and speed (in % total, 100% is fine in most cases) by typing the values into the interface and press the "+" or "-" buttons. If the numbers do not appear upon starting the interface see trouble shooting section for further assistance, this indicates a manipulator error and will require you to restart the program.
6. Automated Microinjection Controls -The trajectory controls are as follows (see section V for parameter selection values): ❖ Approach Distancethe distance the pipette pulls out of the tissue before advancing to next injection site in microns.
❖ Depth -the axial depth into the tissue the pipette goes upon injection in microns.
❖ Spacingthe spacing between subsequent injections in microns.
❖ Speedthe speed of the manipulator in microns/s 7. Microscope Video Stream -The images from the microscope camera are displayed here as a live video stream.

IV. Turning on Devices
Before the experiment turn on the following devices (it does not matter what order you turn on the devices). It is recommended you turn on the devices at least 10 minutes before you intend to do a microinjection.

Turn on the Pressure Rig Switch
a. The pressure rig is shown on the images below. Turn on the power rig by flipping the switch (zoomed in image on right). The image below zoomed in shows the power supply switch in the 'ON' state.

Turn on the camera
a. If you are using the Hamamatsu camera flip the switch shown in the image below. You will hear the fans of the camera start when you turn the camera on. b. The camera USB should always be plugged into a blue "USB 3.0" port like the one on the back of the computer as shown in the image below to the right.

Turn on Pressure Sensor
a. Hold the ON/OFF button to turn on the pressure sensor (displayed in PSI) indicated by the red arrow in the image to the right.

Verify Arduino USB Connection is plugged in
a. The Arduino is indicated by the blue arrow in the image below. Verify the device is connected to the computer via USB. Small LEDS on the Arduino will light up when the Arduino is connected V. Running Experiment 1. Place sample in sample holder on the microscope stage as shown in the left image above.
2. Load pipette with injection medium and mount onto manipulator as shown in the top right image above. Notice how the end of the pipette holder aligns with the end of the sliding stage and is parallel to the axis. Make sure the screw is tight to ensure a stable pipette 3. Load the application by clicking the file "launchapp.py" in the main folder downloaded from github. See github/section III for in depth software description.
First a small black screen will appear followed by the GUI application. This may take up to 30s to load software the first time so be patient. The pop up screen below will show up (left image). Select the options shown in the right image (Camera = Hamamatsu Orca DCAM, com = com5, Res test = off) and click "save and exit". You may need to select a different com port based on your computer (see trouble shooting/github to figure out which com port is appropriate).
4. If the manipulator was loaded correctly you should lines in the system status shown in the image above, and numbers appear in the top right manipulator panel as shown in the top portion of the image to the right. Before submerging the pipette into the solution, it is necessary create outward pressure to prevent unwanted clogging. Slide the compensation pressure to an arbitrary value indicated by red arrow in image to right (24-45% works) and click set values. This will apply pressure to the pipette (you do not have to enter the other parameters yet although they are shown in this image).
5. To obtain desired pressure turn the mechanical knob shown in the image below (clockwise to increase, counter clockwise to decrease). The units are in pound per square inch (1.08 PSI = 75mbar, which is what we use in our experiments for dye, 1.81PSI = 125 mbar for use with mRNA). This can vary widely based on the solution so it is more of a relative value and whatever pressure produces appropriate fluorescence is what you should use.
6. Bring the desired area into focus under the 10x objective and lower the pipette into the solution in this area. You can see that the pipette has been submerged by observing a slight dimple in the water as shown in the image below. Before going lower, search the entire Z area for the pipette using the microscope focus. Once you have found the pipette lower it into the solution in small steps and refocus. Repeat this process until the pipette is in the same focal plane as the tissue.
7. Depending on your microscope, switch the optical output from the microscope to the computer press the middle of the three buttons in the image to the right (shown with the caption "left/right" on the microscope 9. Calibration -(see Movie EV1, and methods for more detail). Click the magnification button in the top left of the interface. A window will prompt you to select the magnification. Select 10x and press 'Ok'.
10. Now, we need to calibrate the Autoinjector relative to the camera axes. Refocus the pipette tip and click the pipette tip with your cursor (number 1 in the image below). A white dot will appear where you clicked. Now, press step 1.1 as shown in the image below (number 2) and press OK in the popup window. The pipette will move in the Y direction.
11. Click the tip of the pipette again (number 3) and press step 1.2 as shown in the image below (number 4). The autoinjector is now calibrated.
12. Now we need to enter the angle between the pipette X axis and the true x axis. This value should not change during operation of the device (it is usually 45.2 -45.4). Thus, you do not need to check it every time. Simply enter "45.3" into the area and press "set angle". It makes sense to check this value once per week to make sure it is not changing. To find this angle follow the following steps using the manipulator interface: 13. If desired, you may test that the calibration has worked well click the "Draw Edge" button as shown in the image to the right. 18. Click "Run Trajectory" button this will start the trajectory. Observe the trajectory of the pipette, if this is satisfactory proceed, if it is not, recalibrate manipulators as described above, or see trouble shooting section. After the trajectory is finished, the number of attempts will be displayed in the bottom system status monitor.
19. Before injection, verify pipette is not clogged. Switch the viewing back to the microscope (see step 8), switch on epi shutter (beneath transhutter from step 6), and flip filter wheel (beneath objectives) to appropiate wavelength. You should see a small cloud of dye being emitted through the tip of the pipette. If you see no cloud, increase the pressure (step 5). If you increase the pressure (above 10 PSI) and see no cloud, the pipette is clogged (see section VI. Trouble shooting).
20. At this point, you may need to reposition the tissue and search for an appropiate focal plane for injection. The ideal tissue area will have an edge that is sharp within the same focal plane.
21. When the ideal focal plane is found adjust the pipette and redraw the desired trajectory by clicking the "draw edge" button, bring the pipette close to the top of the trajectory, and click the tip of the pipette as shown in the image above.

Click "Run Trajectory"
23. If at any point you wish to stop the process, click "Stop Process" which is located beneath the "Run Trajectory" button. The ideal slice will have 1 -4 focal planes for injection and you can repeat step 20 -22.
24. After use, pull pipette away from the slice, remove slice, and reposition stage to next slice if applicable. You do not need to recalibrate the pipette unless you change the pipette. Repeat steps 20 -23 if desired. IMPORTANT, do note pull the pipette out of the solution or it will become clogged.
25. After you have completed injections, remove the pipette from the solution, remove slices, and turn off all devices (order is not important).

VI. Trouble Shooting
See the video tutorials for information on how to troubleshoot specific issues. The following issues are easily solvable. However, if your issue persists or is not on this list feel free to contact us. • Issue -When running trajectory, pipette goes to first location but freezes in place.
Cause -There is an Arduino connection problem. Confirm this by minimizing the interface and looking at the black box window behind the interface. If you see the words "there is most likely an Arduino error" then there is most likely an Arduino error. Solution -Unplug the Arduino USB and replug the USB, if problem persists try another USB port.
• Issue -Camera feed is not displayed, but is replaced with text that says "CAMERA ERROR". Causes -The camera is not turned on, the camera is not properly connected, there is another instance of the application open (two windows of the same app). The wrong software is implemented for the wrong camera. Solution -Verify proper connections are made and device is on, close applications and wait 2 minutes for applications to be killed properly, make sure correct camera was selected from the dropdown menu.
• Issue -Pipette is clogged Causes -molecules aggregate, accidently punctured tissue, pipette is not ideal shape.
Solutionscentrifuge injection solution and remove only supernatant, replace pipette, pull pipette at 2 deg lower, and lower pull value to 50. Elena and Christiane can provide good feedback on this as well.

VII. Guidelines
These guidelines provide user with practical notes for implementing automated microinjection. We limit our discussion to microinjection into single neural stem cells and neurons in tissue. Further optimization may be needed when users are attempting to adapt the Autoinjector for microinjecting other types of tissue or cell types.

Parameter Description Typical Values Troubleshooting
Pipette Shape The shape of the pipette is controlled by the pipette puller parameters and plays a role in minimizing tissue damage and pipette clogging.
-See figure 1 for a picture of a good injection pipette.

Depth of injection
The depth of microinjection should be minimized to prevent cell damage but maximized to increase injection yield.
-It is helpful to vary injection depth and quantify yield as we did in Figure 3 of the main manuscript (varied from 10 -25 µm for APs). As for the parameters of the puller, one should keep in mind that the puller parameters (Time, heat, Velocity, etc) are strongly depending on the puller itself and on the specific ramp test temperature of the pipette one uses. The ramp test must be determined by the user before starting to use the puller. We provide as an example the parameters that we did use for our pipettes, and the corresponding ramp test of the glass capillaries that were used.

The chemical nature of the microinjection solution and its effects on the efficiency of injection
Microinjection relies on the use of pressure to introduce chemicals into cells, as such, one can microinject virtually every compound (and their combination), irrespective of its chemical nature. The success of microinjection can be influenced by the chemical nature of the compound to be microinjected. In particular, the intrinsic tendency of a compound

MICROINJECTION Determining inter-injection spacing
While setting the spacing parameter, one should consider two factors:

Tissue Viability:
Choosing a small spacing (< 10 µm) might have detrimental effects on tissue (and cell) viability due to the increased mechanical stress. In our hands, a minimum of 15 µm spacing was used. This (or larger) spacing was found not to have any noticeable effect on tissue viability and tissue structure. We do not recommend using a smaller spacing. In case the user needs to use a smaller spacing then we do recommend running viability controls to rule out detrimental effects.

Ability to discriminate single cells post injection:
A small spacing (< 10 µm) is going to negatively affect single cell resolution, as cells will be too close to be distinguished unambiguously. We recommend using > 15-20 µm to secure good single cell resolution in case one wants to inject apical progenitors (APs). For injecting neurons, we recommend using > 30 µm, as the neuron structure is normally quite complex and extends on a very large area.