How does 2 photon calcium imaging work?

Two-photon calcium imaging has been widely used to image the activity of neurons in awake behaving animals. Neurons are loaded with a calcium-sensitive dye or, more commonly, made to express a genetically encoded calcium indicator, such that their fluorescence signal reflects spiking activity of the neurons.

What is Vivo 2 photon imaging?

Abstract. Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo. In the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes.

What is calcium imaging used for?

Calcium imaging can be used to track the activity of neurons over time and investigate how networks grow or change during learning. This is especially important for the longitudinal study of animal models.

What is GCaMP6s?

GCaMP6s is a genetically encoded fluorescent Ca[2+] indicator that shows high sensitivity and slow decay kinetics. It consists of the calmodulin-binding peptide M13, a circularly permuted green fluorescent protein and calmodulin.

What are calcium indicators?

Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca2+ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing.

Is calcium imaging electrophysiology?

Calcium imaging using fluorescent protein sensors is a powerful method for recording activity in large neuronal populations[5,8]. In systems neuroscience, cellular calcium imaging fills a complementary role to extracellular electrophysiology.

What are genetically encoded calcium indicators?

The genetically encoded calcium indicator (GECI), which binds to calcium ions and emits fluorescence visualizing intracellular calcium concentration, enables detection of in vivo neuronal firing activity. Various GECIs have been developed and can be chosen for diverse purposes.

What is in vivo fiber photometry?

In vivo fiber photometry is a powerful technique to analyze the dynamics of population neurons during functional study of neuroscience.

What is iGluSnFR?

The iGluSnFR construct provides an improved means to map excitatory synaptic activity in the brain directly and will complement existing imaging methods for studies of neural activity and signaling events.

What is cpGFP?

To visualize Ca2+-dependent protein–protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini.

How does EDTA react with calcium ions?

As a result, when the calcium ion–PR complex is titrated with EDTA the Ca2+ ions react to form a stronger complex with the EDTA. For the titration, the indicator is added to the sample solution containing the calcium ions and forms the pink/red calcium ion-indicator complex (Ca-PR).

How Calcium imaging is done?

Images are analyzed by measuring fluorescence intensity changes for a single wavelength or two wavelengths expressed as a ratio (ratiometric indicators). If necessary, the derived fluorescence intensities and ratios may be plotted against calibrated values for known Ca2+ levels to measure absolute Ca2+ concentrations.