Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

The overall goal of the following experiment is to perform the key aspects of chronic two photon imaging in mice, exploring a virtual reality environment. This is achieved by modifying a two photon microscope for fast and stable imaging in behaving mice. Next, an air supported treadmill capable of locomotion tracking is set up.

Then a virtual reality environment is prepared that uses an LED projector synchronized to the microscope in order to minimize light leak. As a result, good and stable image recordings are obtained due to the reduction of movement artifacts, the reduction of light leak, and the minimization of photo damage edge. Although this method can be used to measure neuroactivity during navigation in a virtual reality environment, it can also be easily adapted to a wide variety of different behavioral paradigms that require visual stimulation.

All animal procedures were approved and carried out in accordance with the guidelines of the veterinary department of the Canton Basil sta. The two photon microscope used in these experiments consists of a scan head composed of an eight or 12 kilohertz resonant scanner and a standard galvanometer. This enables frame rates of 40 or 60 hertz at 750 times 400 pixels and reduces brain motion induced image distortion, photobleaching and phototoxicity.

A piezoelectric high speed Z stepper is used to sequentially acquire time interleaved images at multiple depths. A mechanical blanker is used to reduce laser exposure to the tissue at the turnaround points of the scan path, the blanker has to be adjustable to set its width. Depending on the amplitude of the resonant scanner, connect an air supply tube with at least a 10 millimeter inner diameter to the ball holder of the treadmill and place an BLE amount of tubing between the main air supply and the ball holder to reduce noise.

Place a polystyrene foam ball, 20 centimeters in diameter in a custom designed and 3D printed ball holder. If the behavioral paradigm only requires forward and backward locomotion, insert a pin such as a 19 gauge hypodermic needle on the side of the ball. To restrict movement of the ball in the horizontal axis, use one or two optical computer mice to track movement of the ball around two or all three axes respectively.

To prepare the virtual reality environment, use LED projectors or LED backlit computer screens for the display. Integrate a gating circuit for fast flickering of the LEDs during imaging. The 24 kilohertz flickering frequency is orders of magnitude above the flicker fusion threshold, above which flickering is no longer perceived by the animal.

Adjust the exact flickering frequency and duty cycle to synchronize the light output of the projector to the turnaround points of the resonant scanner for pupil tracking, use a CMOs based video camera. Make sure that the camera does not contain an infrared blocking filter. Approximately two to three weeks after injecting the genetic calcium indicator and implanting a cranial window, use epi fluorescence illumination to check the cranial window implant for clarity and expression.

Clearly visible and sharply defined. Boundaries of superficial blood vessels are a good indication that the window implant is suitable for imaging one to two days later. To carry out the two photon experiment, measure and adjust the laser power to a value below 50 milliwatts at the sample.

Then turn on the airflow to the spherical treadmill. Next head, fix the animal on the spherical treadmill. The first few times the mouse is head fixed.

This process can be eased by briefly sedating the animal with iso fluorine. Once the animal has become accustomed to the procedure, mounting can normally be performed without sedation. Note that it is critical to minimize stress to the animal during this procedure.

Then position the visual display system as close as possible to the animal to still allow for access to the animal. Make sure the cranial window implant is free of dirt and clean it with water if necessary, apply clear ultrasound gel as immersion medium between the cranial window and water immersion objective for light shielding. Wrap a piece of black tape around the objective and head bar.

Move the virtual environment arena to its final position and adjust the video camera for pupil tracking. Finally, set the laser blanker to match the scan amplitude used during the experiment and start recording data when experiments are complete. Euthanize the animal according to your institution's guidelines.

The image quality in two photon calcium imaging of cell populations labeled with a genetic calcium indicator largely depends on the quality of the cranial window implant as demonstrated here when checking the cranial window for clarity after virus injection, there should be no granulation tissue or bone regrowth visible. Moreover, the pattern of superficial blood vessels should remain unchanged and the boundaries of the vasculature should be sharply defined. At the same time, BOI of labeled cells should be clearly visible under an epi fluorescence microscope.

Ideally, the preparation is stable enough such that distortions of the two photon images induced by locomotion of the mouse are not visible in the image after frame registration. Light leak from the visual stimulation system can be fully abolished by correctly timing the light output of the projector to the turnaround points of the resonance scanner. When data are not acquired, the flickering frequency of the light source should be twice the scan frequency.

In this example, the blanker was removed to illustrate the effect of light leak shown in the orange boxes during visual stimulation. After watching this video, you should have a good understanding of how to perform twofold in imaging, behaving mice, navigating a virtual reality environment. In addition to setting up the imaging and virtual reality systems as described here, the key to success in these experiments is mastering the cranial window implant technique.

A clean implant is a prerequisite for high quality images and stable recordings.