Single-cell two-photon calcium imaging

Imaging at a single-cell level is a powerful technique. Not only will it allow us to visualise the morphology of a neuron down to its dendritic spines at a high spatial resolutionl and measure its calcium transient but we can also look at how input signaling works. This is useful in giving light to questions such as what are the changes and impact in signaling between axons of normal and abnormal cells. It is fundamental to answer such questions if we are to understand how the input and output signal, let’s say the communication, between neurons at a cellular level works.

I wanted to highlight here the technical method in terms of its preparation and the delivery using a two-photon (2P) microscope. The success of this type of experiment highly depends on the success of craniotomy and the delivery of the synthetic dye, oregan green BAPTA-1 hexapotassium salt (OGB-1). Even after lots of practice, it will take several months and up to one year to really master the art of craniotomy. Things can be pretty tricky therefore, it is important to keep the animal in a good physical status during the experiment. To do this, the temperature and its heart rate must be monitored at all times. Typically it should be at 37 oC between 60-80 bpm (beats per min) during the craniotomy (2-3 mm), respectively. Its heart rate was brought up to 100-120 bpm during the experiment by increasing the flow of isoflurane.

The above is an example of a typical 3D stack of a single cell electroporation. We did this by loading the OGB-1 (9 μM, 5 μL) into the pipette. The targeting pipette tip, angled at 45 degree is guided to the desired location using the 2P for visualisation. Leaks from the pipette is usually enough to enable visualisation under the tissue and it is crucial to avoid the any blood vessels. A neuron is labelled by firing a single pulse of 50 ms with an electrical field of 2.4 ps. This is enough to permeabilise the membrane of the cell in order to deliver the OGB-1. It is best to image after 30 – 60 mins after the electroporation. This is to make sure that the cells are brought back up to its normal physiological condition. If the neuron is healthy, its dendrites should look uninterrupted. A blur of fluorescence could indicate that they are burnt from the laser. If time is not a limiting factor, the ultimate test is to look at its transient or spike activity. It’s pretty cool and it can be very tricky at this point. It’s like a video game when you have to aim and shoot perfectly. It is a very lengthy process when things start to fail. Like when the animal stops breathing, blood vessels bursts, the dye diffuses out of the pipette and so on.

Here, the calcium transient in-vivo imaging was easily observed even along the dendrites and its dendritic spines. I got the chance to play around with the ImageJ. It was used to analyse stack projections of the experiments with a 1 μm between each plane. Getting to this point requires experience, practice and more practice. Perhaps you are thinking of using this technique and my advice is to read up on papers on these methods. The most useful ones will be referenced here. The discussions about this technique are very useful for troubleshooting and therefore highly encourage.

[1] Chen, X., Leischner, U., Rochefort, N. L., Nelken, I. & Konnerth, A. Functional mapping of single spines in cortical neurons in vivo. Nature 475, 501–5 (2011).

© 2014 So you think you can grow crystals in a beaker.

* Experiments demo sessions, 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”.

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