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Calcium imaging is a biological method used in neuroscience for visualizing the activity of neural circuits in the brain. By detecting changes in fluorescence due to fluctuating calcium, calcium imaging is an indirect way of measuring neuron activity in freely-behaving animals, head-fixed animals, and in vitro/ex vivo preparations.

Low-cost one-photon microscopy, such as miniscopes or epifluorescence microscopy, is frequently used for calcium imaging experiments. Commonly, the calcium indicator GCaMP is employed for one-photon calcium imaging experiments with high popularity, optimization, and ability to detect single action potentials.

Due to the nature of one-photon imaging and the expression of GCaMP throughout the entire neuron, a major pitfall of one-photon GCaMP imaging is high background fluorescence. The high noise and crosstalk produced by this background can, importantly, lead to potential misinterpretation of imaging data. 

Optically, the issues associated with one-photon GCaMP imaging can be resolved by using two-photon imaging; however, two-photon imaging can be quite costly. 

This begs the question: How can one-photon GCaMP imaging be optimized from the biology side? 

In a recent preprint published by Or Shemesh, Edward Boyden, and colleagues, they focused on producing a soma-targeted GCaMP to decrease crosstalk and provide better quality one-photon GCaMP imaging.

What Did the Authors Find?

Comparison of fluorescent calcium sensor expression for GCaMP and soma-targeted GCaMP.

In this study, GCaMP6f was fused with different proteins known to express somatically in the cell. These GCaMP6f variants were expressed in culture and slice to test for localization, toxicity, and levels of expression. Derived from these exploratory methods, there were two variants of soma-targeted GCaMP6f found: SomaGCaMP6f1 and SomaGCaMP6f2. These two variants show localized expression in the soma, detected single action potentials, and displayed similar signal-to-noise ratio to GCaMP6f. 

Using electrophysiological recordings in slice, SomaGCaMP6f1 detected single action potentials at higher frequencies more accurately than GCaMP6f. These findings were similarly seen using one-photon light-sheet microscopy, when SomaGCaMP6f1 was expressed in the Zebrafish brain.

Lastly, SomaGCaMP6f1 and SomaGCaMP6f2 were tested in vivo by expressing these constructs in the mouse dorsal striatum. Compared to GCaMP6f, SomaGCaMP6f2 displayed similar brightness levels; however, SomaGCaMP6f1 showed much lower levels of brightness in vivo. Thus, one-photon imaging was performed on a head-fixed mouse running on a treadmill expressing either SomaGCaMP6f2 or GCaMP6f. SomaGCaMP6f2 imaging led to greater soma-localized responses and detected more calcium events, compared to GCaMP6f.

Take-Away

Shemesh and colleagues demonstrate the viability of using these newly developed soma-localized GCaMP variants to improve background and crosstalk associated with one-photon calcium imaging. Hopefully, the continued refinement of biological constructs for calcium imaging can help improve the acquisition and analysis of one-photon calcium imaging experiments

To read the original preprint, see below.

Or, A et al. Precision calcium imaging of dense neural populations via cell body-targeted calcium indicator. Biorxiv.

 

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