This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.
Fiber photometry CSOM imaging combines genetically encoded custom indicator and optical fibers to monitor neural activity in freely-moving animal. Which is critical to understand how a specific group of neurons play in directing or responding to an action or a stimulus. This technique is an accessible way to record from specific brain regions that are defined by their connection or genetic profiles, with the built in control to separate signal to noise.
Demonstrating the processor will be, Ekaterina Martianova, a graduate student in my lab. Begin by loosening all screws on the five axis translator. Screw in the patch cord to the adaptor that is affixed to the five axis translator.
Then turn on the 470 nanometer excitation light at low power and position the tip of the fiber pointing to an auto fluorescent plastic slide. Next record from the complimentary metal-oxide semiconductor or CMOS camera in live mode. Increase the gain or adjust the lookup table until the image is visible at the focal point of the objective.
Advance the five axis translator towards the objective ensuring that the 470 nanometer light is centered on the fiber at the SMA or FC end of the patch cord, until an image can be resolved on the camera. Finally, adjust the X and Y axes until the image is centered and well resolved. Visualize the light emitted from the variole end of the patch cord, as it should appear as an isotropic circle.
Start by turning on all the excitation lights to better visualize the fibers. And adjust the camera gains such that no pixels are saturated, and a clear image of the fibers are present. Then take a preliminary image.
Draw regions of interest or ROIS around the fibers. And keep them for the measurement of the mean intensity values during recordings. For multiple fiber recordings, test for independents by live recording from all fibers.
Point one fiber towards a light source, and tap with a finger. Observe fluctuations in the channel. Then apply colored tape to the end of the fibers to label which ROI corresponds to which fiber.
Finally, set up the recording area by hanging the patch cord above the arena using stands, clamps, and holders. Lastly, make sure that the animal will not be limited in movement by the length of the fiber and can freely move throughout the entire arena. Begin by inspecting the distal end of the fibers of the patch cord by eye, and with a mini fiber microscope.
If the surface of the fibers is scratched, re-polish the fibers using fiber polishing film with fine grit. Then clean the distal ends of the patch cord with 70%ethanol and a cotton tip applicator. Clean the fiber optic cannulas using 70%ethanol and a cotton tip applicator.
Connect the variole end of the patch cord to the implanted fiber using a ceramic split sleeve covered with a black shrink tube. During the connection, make sure that the sleeve is tight. Allow the animal to recover for a few minutes.
Then start recording the optical signal, and run the experiment. While recording keep a careful eye on the live trace to ensure quality recordings. And watch for any jumps in signal, indicating the sleeve is not tight enough.
For the dual color recordings, add a 516 nanometer LED to the photometry system to excite the red fluorescent calcium sensor, and appropriate dichroic mirrors and filters. Then add an image splitter between the objective and the CMOS camera to separate the green and red emission wave lengths. Finally, draw ROIS around all fibers in both colors, red and green.
Make sure to clearly identify each ROI with the corresponding fiber and channel. Trigger simultaneous excitation with 470 nanometer, and 560 nanometer LEDs, and alternate them with a 410 nanometer LED. Results indicate, the measured fluorescence significantly increased concurrently with the administration of air puffs for mice with activation of the LHA-LHB pathway.
However, in mice expressing green fluorescent protein or GFP, no change in the signal was detected during the administration of air puffs. This technique allows researchers to reasonably record from specific type of cells in different brain regions. While an animal is freely behaving.
This furthers the ability to dissect the functions of these brain regions as they relate to behavior.
