TITLE: Protocol for Neuronal Live-imaging of primary cultures
AUTHORS: Camila Pulido, Timothy A. Ryan

[DESCRIPTION]
Neurons stand out as remarkably intricate cells. The development of live-imaging techniques on primary neurons expressing genetically engineered biosensor has proven pivotal in advancing our comprehension of neuronal function at both cellular
and molecular level. This protocol introduces a concise method for live-imaging neuronal activity.

[BEFORE_START]
Turn on live-imaging setup: Lasers, microscope, mercury arc lamp, stimulus isolator, XYZ stage, imaging computer.
Open acquisition and controller programs (i.e. camera program, laser’s controller, ImageJ).
Make sure the EMCCD camera has reach the working temperature (~ -80°C) before start imaging. 

[GUIDELINES]
The animals should be used in accordance with protocols approved by national and institutional regulatory organizations.

[STEPS]
SECTION: Mounting the coverslip to recording chamber
1.   
Add 2 mL of ‘Imaging Solution’ into a P35 dish.

SECTION: Mounting the coverslip to recording chamber
2. Cut a piece of a new coverslip and fix it with some grease into the ‘Stimulator insert’ (Figure 1).  
 
 
SECTION: Mounting the coverslip to recording chamber
3. Add a drop of grease to every corner (bottom face) of the ‘Stimulator insert’ (Figure 2). 

 
SECTION: Mounting the coverslip to recording chamber
4. Add grease around the circle (facing up) of the ‘platform chamber’ (Figure 3).

 
SECTION: Mounting the coverslip to recording chamber
5. Take a DIV14 – DIV18 neuronal cultured dish from the incubator.
SECTION: Mounting the coverslip to recording chamber
6. Assess for cell density, health and overall conditions using a basic light transmission microscope to ensure the suitability of the dish before starting the experiment.
SECTION: Mounting the coverslip to recording chamber
7. Place the coverslip containing platted cells into the petri dish prepared at  , and leave it for 10 s  to 20 s to equilibrate with the ‘Imaging Solution’.

SECTION: Mounting the coverslip to recording chamber
8. Attach the coverslip to the ‘Stimulator insert’; with the help of some tweezers make sure every corner of the ‘Stimulator insert’ is well attached to the coverslip by the grease.
SECTION: Mounting the coverslip to recording chamber
9. Seal the ‘Stimulator insert’ to the ‘platform chamber’, clean any excess of grease that might be around and make sure there is no leak.

 
SECTION: Mounting the coverslip to recording chamber
10. Clean the bottom of the coverslip with a wipe with  , making it ready for imaging.

SECTION: Checking for transfected neurons under the microscope
11. Place the ‘recording chamber’ into the microscope’s stage.
SECTION: Checking for transfected neurons under the microscope
12. If electrically stimulating neurons, connect the ‘recording chamber’ to the stimulus isolator.
SECTION: Checking for transfected neurons under the microscope
13. Start prefunding ‘Imaging Solution’ trough the coverslip at a speed  of ~100 uL min-1. Make sure all perfusion lines with additional test solutions are calibrated to run at the same speed. 

SECTION: Checking for transfected neurons under the microscope
14. Using transmission light find the focal plane where neurons are located.
SECTION: Checking for transfected neurons under the microscope
15. Change microscope illumination to the mercury arc lamp, making sure the desire dichroic filter is in place. (i.e. GFP-filter cube).
SECTION: Checking for transfected neurons under the microscope
16. Verify the presence of transfected neurons based on their baseline fluorescence emission. If the biosensor’s baseline fluorescence is insufficiently bright due to its inherent nature (i.e. vGLUT-pH), add a suitable solution (i.e. NH4Cl-Tyrode’s) into the chamber that may enhance the biosensor signal, facilitating the identification of transfected neurons.
SECTION: Checking for transfected neurons under the microscope
17. Save all localizations where transfected neurons are found.
SECTION: Checking for transfected neurons under the microscope
18. Proceed with the imaging protocol.
SECTION: Neuronal Imaging Protocol
19. Flip the microscope mirror from the mercury arc lamp towards the laser path.
SECTION: Neuronal Imaging Protocol
20. Make sure the following programs are open at the ‘Imaging-setup computer’: EMCCD camera control software (like Andor-Solis program), laser control software (like Coherent connection), master control program (like a custom-designed Arduino controller program) and image analysis program (like ImageJ with Time-Analyzer plugin).
SECTION: Neuronal Imaging Protocol
21. Load the desired acquisition settings protocol for imaging in the EMCCD camera control software.
SECTION: Neuronal Imaging Protocol
22. Select one of the previously saved stage localizations, re-focus and move the neuron/boutons to a desire field of view, ready to be imaged.
SECTION: Neuronal Imaging Protocol
23. If electrically stimulating, make sure that the correct settings are selected (i.e. number of pulses, stimulation frequency, time to first pulse) and ready to Go when triggered by camera action. 
SECTION: Neuronal Imaging Protocol
24. When ready for imaging, proceed to record.  
SECTION: Additional notes
25. This live-imaging protocol can be adapted to be used with ratiometric biosensors (i.e. iATPSnFR2-HALO), by using the appropriate dual filter cube corresponding to the different fluorescent emissions and, by switching lasers between frames when image acquisition is performed.