There are many techniques for calcium imaging. It is an essential and well-established technique as an indirect method to “see” neuronal activity events in the brain.
In this section, the aim is to demonstrate the capability of optical fibre technique for imaging. We successfully inject the calcium sensitive dye, oregan green BAPTA-1 AM into the superficial layers II/III layers of the visual cortex at a depth of around (100-200 μm) and using the optical fibre system with excitation capability of 450 nm to record the calcium signal of a small population of labeled neurons. Such systems can be set up to interrogate a couple of regions of the brain, such as how does one population of neurons communicate with another population of neurons at a desired region. One would need two fibres running at the same time but you cannot really do this with any other system at such a high temporal resolution.
The graphs I am showing here is a typical raw data of a fibre optic system. What you see is a spontaneous activity of the neurons against recorded as a function of time. The rise in the background noise is due to the fact that OGB-1 AM could still be taken up by the neurons and therefore the baseline fluorescence is increasing. This would be normally corrected for by subtracting the baseline signal. The baseline signal can be calculated in a number of ways, for example, by taking the mean values as certain timepoints and then average all these values. The normalised data is often shown as the ΔF/F0.
The second image is typical of recording of the spontaneuous activity you see when the isoflurane (2% in O2) anaesthesia is increase, in our case from 1.2 % to 1.5 %. Here, the frequency of the overall calcium transient is decreased. This is useful as it gives us more time and control between the baseline and the spontaneous activity for stimulations.
The surgical procedure
A wild type adult C57BL/6J mice were used and was anaesthetized with iso-flurane during the entire surgical procedure. For the initial anaethestic, the animal was exposed to a 2.5 % iso-flurane in pure O2 running at 500 cm3/min for 5 minutes in a closed environment. It was reduced down to <1.5 % during the experiment. It was fixed onto the stereotaxic apparatus on top of a warming plate (37 oC) with the ear bars gently fixed throughout the procedure. Once it was properly placed, the mouse was checked to make sure it was properly anaesthetize by pinching the tail and ear and checking for reflexes, the heart rate was monitored at around 90-100 bearts/min. Its head was exposed to the skull, the objective point on the skull was located and marked the objective point on the surface of the skull. The skull was opened with a diameter of 300-500 um. OGB1-AM was then placed into a specific glass micropipette (500nl, 500 uM), and found the landmark where the hole was drilled. With the stereotatic apparatus, set the location to zero and go down up to 200 um. We first injected a small dose of around 50-70 nL into the superficial layers of II/III of the visual cortex by air pressure. Once this was done, we made sure and waited further 30 mins until the dye was distributed until the pipette was raised back up. Once satisfied, we start the recording by using the optical fibre to the stereotatic holder and descended to the surface of the injection site.
© 2014 So you think you can grow crystals in a beaker.
* Experiments were carried out under the institutional guidelines of the Third Military Medical University, Chongqing as part of the Sino-German Summer Course “two-photon functional imaging of the living brain” as part of a demo session.
Roberts Lab 